Self-assembled monolayers probed by sensorsmonolayer surfaces. First, the design and characterization of several novel β-cyclodextrin (β-CD) adsorbates is presented (Chapters 3 and

Self-assembled monolayers probed by

electrochemistry: From layer properties to

sensors

Graduation committee:

Prof. dr. ir. J.W.M. Hilgenkamp University of Twente (chairman)

Prof. dr. ir. J. Huskens University of Twente (supervisor)

Prof. dr. ir. P. Jonkheijm University of Twente (supervisor)

Prof. dr. J.J.L.M. Cornelissen University of Twente

Prof. dr. S.J.G. Lemay University of Twente

Prof. dr. M. Sollogoub Université Pierre et Marie Curie

Prof. dr. W.R. Browne University of Groningen

Dr. B.A. Boukamp University of Twente

The research described in this thesis was performed within the laboratories of

the Molecular Nanofabrication (MnF) group, the MESA+ Institute for

Nanotechnology, and the Department of Science and Technology of the

University of Twente. This research was supported by the European Research

Council (ERC) through the ERC advanced grant ‘Elab4life’.

ISBN: 978-90-365-4349-1

DOI: 10.3990/1.9789036543491

Printed by: Gildeprint – The Netherlands

Cover art: Lindy Steentjes

Copyright © Tom Steentjes, Enschede, 2017.

All rights reserved. No part of this work may be reproduced by print, photocopy

or any other means without prior permission in writing from the author

SELF-ASSEMBLED MONOLAYERS PROBED

BY ELECTROCHEMISTRY: FROM LAYER

PROPERTIES TO SENSORS

PROEFSCHRIFT

ter verkrijging van

de graad van doctor aan de Universiteit Twente,

op gezag van de rector magnificus,

prof. dr. T.T.M. Palsma,

volgens het besluit van het College voor Promoties

in het openbaar te verdedigen

op donderdag 29 juni 2017 om 14.45 uur

door

Tom Steentjes

geboren op 18 oktober 1982

te Doetinchem

Dit proefschrift is goedgekeurd door:

Promotoren: Prof. dr. ir. J. Huskens

Prof. dr. ir. P. Jonkheijm

Table of contents

Chapter 1: General introduction 1

1.1 References 2

Chapter 2: Electrode transfer on electrode surfaces and applications in

biosensing 3

2.1 Introduction 4

2.2 Electrochemical techniques 4

2.2.1 Chronoamperometry 4

2.2.2 Cyclic voltammetry 5

2.2.3 Electrochemical impedance spectroscopy 10

2.3 Electrochemical DNA sensing 12

2.4 Conclusions 15

2.5 References 16

Chapter 3: Self-assembled monolayers on gold of β-cyclodextrin adsorbates

with different anchoring groups 21

3.1 Introduction 22

3.2 Results 23

3.3 Discussion 34

3.4 Conclusions 37

3.5 Experimental section 37

3.5.1 Materials 37

3.5.2 Synthesis of new adsorbates 38

3.5.3 Methods 45

3.6 References 47

Chapter 4: Electron transfer rates in host-guest assemblies at β-cyclodextrin

monolayers 53

4.1 Introduction 54

4.2 Results and discussion 55

4.3 Conclusions 71


4.5 References 79

Chapter 5: Electron transfer processes in ferrocene-modified poly(ethylene

glycol) monolayers on electrodes 85

5.1 Introduction 86


5.3 Conclusions 93


5.4.1 Materials 94

5.4.2 Surface functionalization and electrochemistry 95

5.4.3 Calculations 96

5.5 References 96

Chapter 6: Electron transfer mediated by surface-tethered redox groups in

nanofluidic devices 99

6.1 Introduction 100


6.3 Conclusions 109


6.5 References 113

Summary 117

Samenvatting 119

Acknowledgements 121

Curriculum Vitae 123

1

Chapter 1

General introduction

Electron transfer processes are of importance in a broad range of fields, such as

biosensing1 and molecular electronics.2 Gold electrodes can be readily

functionalized using thiol chemistry3 for the formation of self-assembled

monolayers (SAMs). SAMs constitute an excellent platform for sensing because

of the possibilities to precisely tune the surface composition and the distance

from the electrode surface. As such the influence of the electron transfer

properties of a redox probe that is present in solution or attached at the SAM

can be tuned.

Electrochemistry utilizes redox couples in solution, where the electron transfer

is mostly dictated by diffusion, and attached to a surface where diffusion either

plays no role or is mediated via a flexible linker. When the redox couple is in

solution it can be used to probe the electron transfer through a layer, and the

layer can be designed to block the electron transfer as an insulating layer, or to

optimize electron transfer for the use in molecular electronics.

The work described in this thesis aims to further our understanding of the design

and study of the electron transfer properties of two different types of

monolayer surfaces. First, the design and characterization of several novel β-

cyclodextrin (β-CD) adsorbates is presented (Chapters 3 and 4) with the aim to

study the influence of the distance of the β-CD core to the electrode surface on

the electron transfer properties using different redox probes. Secondly, layers

of linear flexible poly(ethylene glycol) (PEG) polymers end-tagged with an

electrochemically active redox group are described in Chapters 5 and 6 to study

the effect of polymer length and conformation on the electron transfer

characteristics, both on macroscopic electrodes as well as in nanofluidic devices.

Chapter 2 provides an overview of several common electrochemical methods

and looks into their uses for the determination of the electron transfer rate

constants. A further focus is on flexible probes such as DNA and their use in

electrochemical biosensing devices that provide a signal upon changing the

linker conformation.

Chapter 1

2

Chapter 3 describes the design and characterization of six multivalent β-CD

adsorbates that provide close contact of the β-CD cavity to the gold surface.

Monolayers of these β-CD adsorbates were characterized and the adsorption

kinetics, thickness, layer stability and number of anchoring groups bound to the

surface were assessed for the different anchoring groups.

In the work described in Chapter 4, monolayers of four of the β-CD adsorbates

introduced in Chapter 3 were further characterized with regard to their packing

densities using electrochemical methods. The electron transfer kinetics was

studied using different redox probes, both in solution and reversibly

immobilized on the host monolayers. Electron transfer rates between the novel

adsorbates in close proximity to the surface were compared to that of a β-CD

adsorbate further away from the surface by long thioether chains.

Chapter 5 describes the electron transfer for linear PEG polymers attached to

the Au electrode surface on one end and tagged with a ferrocene moiety on the

other end. The effects of bounded diffusion on the electron transfer was studied

and compared for different polymer lengths, together with the effect of

conformational changes as a function of the surface density.

In Chapter 6, the molecules described in Chapter 5 were introduced into

nanofluidic devices with nanospaced electrodes. The effect of surface-bound

diffusion of the ferrocene probe on the repeated shuttling of electrons between

the two electrodes was studied. Subsequently, the effect of linker length and

surface density was assessed in order to validate the potential for using such a

device architecture for biosensor applications.

1.1 References

(1) Labib, M.; Sargent, E. H.; Kelley, S. O., Electrochemical Methods for the Analysis of

Clinically Relevant Biomolecules. Chem. Rev. 2016, 116, 9001-9090.

(2) Adams, D. M.; Brus, L.; Chidsey, C. E. D.; Creager, S.; Creutz, C.; Kagan, C. R.; Kamat,

P. V.; Lieberman, M.; Lindsay, S.; Marcus, R. A.; Metzger, R. M.; Michel-Beyerle, M. E.;

Miller, J. R.; Newton, M. D.; Rolison, D. R.; Sankey, O.; Schanze, K. S.; Yardley, J.; Zhu, X.,

Charge Transfer on the Nanoscale: Current Status. J. Phys. Chem. B 2003, 107, 6668-

6697.

(3) Yildiz, I.; Raymo, F. M.; Lamberto, M., Self-Assembled Monolayers and Multilayers of

Electroactive Thiols. In Electrochemistry of Functional Supramolecular Systems, John

Wiley & Sons, Inc.: 2010; pp 185-200.

3

Chapter 2

Electron transfer on electrode surfaces and

applications in biosensing

The increasing demand for diagnostic tools in personalized medicine calls for an

increase in sensitivity and specificity for the detection devices that are developed

to this purpose. Electrochemistry provides a range of tools which can meet these

requirements. Metallic electrodes can be readily functionalized using thiol

chemistry and provide an excellent basis for tuning the electrode surface for

specific goals. This chapter reviews several common electrochemical techniques

such as chronoamperometry, cyclic voltammetry and electrochemical

impedance spectroscopy, and their uses for the determination of electron

transfer rate constants. Redox-active moieties attached to long flexible linkers

form a special group as their electron transfer partly relies on diffusion. The

conformation of these linkers can be modified by changing the surface density,

and in the case of DNA the stiffness can be affected upon binding with the

complementary DNA chain. These strategies have been utilized for the

development of biosensing devices such as E-DNA and aptamer-based detection

schemes.

Chapter 2

4

2.1 Introduction

Electron transfer processes play an important role in a wide range of fields, from

molecular electronics1, to the development of biosensors.2-4 The growing

interest in personalized medicine and point-of-care testing demands an increase

in sensitivity, specificity, miniaturization, affordability and ease of signal

interpretation.5 Several of these requirements are met by electrochemical

devices.6 As devices become smaller, chemical modification of electrodes

provides an excellent way for control and tunabillity of the chemistry of the

detection probe.7

Electrochemistry is suited for sensitive and accurate measuring of molecular

redox processes both in solution and at the surface. This chapter will focus on

electrochemical processes on electrode surfaces, specifically with the redox

probe covalently attached to the surface via the formation of self-assembled

monolayers (SAMs) to eliminate the use of additional probes to the analyte

solution. The affinity of thiols to noble metals is well known as a way to attach

(electroactive) molecules to surfaces.8

In the first part of this chapter, several commonly used electrochemical

techniques will be discussed together with their uses for the determination of

electron transfer kinetics, with a focus on ferrocene alkylthiols as a key example.

The electron transfer plays a central part in the detection mechanism of

biosensors as they rely on conformational changes upon detection. In the

second part, E-DNA2, 9 and aptamer-based sensors2, 10-11 rely on this principle and

will be discussed in further depth.

2.2 Electrochemical techniques

A redox-active SAM in its most basic form is a redox couple immobilized at, but

separated from, the electrode by a short linker. The affinity of thiols for noble

metals such as gold can be exploited for functionalization of the electrode.

Electrochemical methods provide powerful tools for the characterization of

these types of layers. A plethora of electrochemical techniques is available, a

few of the more commonly used techniques will be discussed here.

2.2.1 Chronoamperometry

In chronoamperometry the potential is changed step-wise and the recorded

current follows an exponential decay according to equation 2.1:12

Electron transfer on electrode surfaces and applications in biosensing

5

𝑖(𝑡) = 𝑘𝑄𝑒−𝑘𝑡 (Equation 2.1)

When the natural logarithm of the current i is plotted versus the time, the slope

can be used to determine the rate constant k. In addition, this analysis can be

performed at varying overpotentials giving a more complete picture of the

influence of the potential on the kinetics as predicted by the Butler-Volmer

behavior. The Butler-Volmer formulation states that the electrode kinetics at an

electrode depends on the overpotential, E - E0’, as shown in equation 2.2, giving

rise to the characteristic Tafel plot.12

𝑘f + 𝑘b = 𝑘0exp(−𝛼𝑅(𝐸 − 𝐸0′)/𝑇) + 𝑘0exp((1 − 𝛼)𝑅(𝐸 − 𝐸0

′)/𝑇)

(Equation 2.2)

Where kf and kb are the forward and backward rate constants, k0 the standard

heterogeneous rate constant, R the gas constant, T the absolute temperature, E

the electrode potential, E0’ the formal potential of the electroactive group and

α the transfer coefficient.

Chidsey was the first to use this method on SAMs of alkanethiols modified with

ferrocene.13 These SAMs were made by mixing the ferrocene alkylthiols with

electrochemically inactive alkylthiols to sufficiently space the ferrocene

moieties and prevent cross-interactions. It was shown that for low

overpotentials the Butler-Volmer formulation was followed, but curvature far

from E0’ could not be accounted for with these formulations. This was overcome

by fitting the data with a prefactor for electron transfer and the metallic states,

and λ, the reorganization energy, i.e., the energy needed to distort the atomic

positions of the reactant and its solvation shell to the atomic positions of the

product and its solvation shell. More importantly, it showed the possibility of

these layers to be probed systematically on the dependence of distance,

medium and spacer structure.13 A wide variety of studies for different alkane

chain lengths have been performed to determine the electron transfer kinetics

using different techniques, and these have been summarized by Eckermann et

al.7 These layers will be discussed in more detail below.

2.2.2 Cyclic voltammetry

In cyclic voltammetry, the potential at an electrode is varied linearly with time

while simultaneously the current is recorded. Typically, the responses of

electrochemically active layers that are surface confined are different than

those of diffusion-controlled processes,12 and the former show a linear

Chapter 2

6

dependence of the peak current (Ip) on the scan rate (ν) (Equation 2.3). The

charge (Q) under the recorded anodic and cathodic peaks is proportional to the

number of redox centers contributing to the electron transfer, and consequently

the surface density (Γ) can be determined (Equation 2.4).

𝑖p =𝑛2𝐹2

4𝑅𝑇𝜈𝐴𝛤 (Equation 2.3)

𝑄 = 𝑛𝐹𝐴𝛤 (Equation 2.4)

Here, n is the number of electrons involved in the electron transfer process, F

the Faraday constant, R the gas constant, T the temperature and A the surface

area of the electrode. The electron transfer of surface-confined electrochemical

species has been described by Laviron14 and the rate constant for electron

transfer (kET) can be determined from the change in peak potential (Ep) with

increasing scan rate. When the difference between the anodic and cathodic

peak potentials exceeds 200 mV, the electron transfer is said to be

electrochemically irreversible and the peak potentials change linearly with the

logarithm of the scan rate. At the intercept of the x-axis of this linear change,

the scan rates va and vc can be determined and the kET can be determined from

Equation 2.5:

𝑘𝐸T =𝛼𝑛𝐹𝜈c

𝑅𝑇=

(1−𝛼)𝑛𝐹𝜈a

𝑅𝑇 (Equation 2.5)

The electron transfer in ferrocene alkylthiols has been studied using a variety of

methods, as mentioned earlier.7 Cyclic voltammetric measurements on different

lengths of alkylthiols at low temperatures have shown that the electron transfer

in well-packed SAMs drops exponentially according to equation 2.6.15 This effect

had been previously shown for pentaaminecobalt(III) anchored to gold and

indirectly reduced via Ru(NH3)62+.16

𝑘ET(𝑥) = 𝑘ET(𝑥 = 0)𝑒−𝛽𝑥 (Equation 2.6)

Where x is the distance to the surface and β expresses the overall reaction rate

sensitivity to distance. Cyclic voltammetry proved to be more suitable than

potential step methods as it is not as susceptible to the effects of kinetic

dispersion caused by structural disorder in the monolayer.17

Since Chidsey’s first work on ferrocene alkylthiols on electrodes18 these layers

have been studied widely. Ideal electrochemical behavior is found when these

SAMs are diluted with electrochemically inactive alkylthiols. At high ferrocene


7

densities significant peak broadening and peak splitting has been observed.18-19

It was found that the peak splitting observed in cyclic voltammetry was

consistent with local effects expected from phase separation, and the two peaks

could be attributed to ferrocene moieties surrounded by neutral alkylthiols

(peak I in Figure 2.1b), or by other ferrocene species (peak II in Figure 2.1b).20

This property has been utilized in order to determine the homogeneity of

alkylthiol layers by exposing formed SAMs to a ferrocene alkylthiol solution

providing access to different types and populations of defects. Single site defects

would only be filled with one ferrocene alkylthiol molecule (Figure 2.1a), i.e.

surrounded by neutral alkylthiols (corresponding to peak I), whereas collapsed

site and pinhole defects provided room for multiple molecules, giving rise to the

faradaic signal corresponding to ferrocene moieties surrounded by other

ferrocene species (corresponding to peak II).21

Figure 2.1: a) Three different types of defects probed by backfilling with a ferrocene alkylthiol,

from left to right a single site defect, collapsed defect and a pinhole. Reproduced from Ref. 21

with permission from the PCCP Owner Societies. b) Cyclic voltammetric response of ferrocene

alkanethiols on a gold electrode for different solution ratios of electrochemically inactive (C10SH)

and ferrocene tethered alkylthiols (FcC12SH). Reprinted with permission from Ref. 20. Copyright

2006 American Chemical Society.

When the kinetics of the electroactive group is controlled by diffusion, the

Laviron method is not suitable for the determination of the kinetics. Typically,

diffusion-controlled electron transfer applies to species in solution, but diffusion

control has also been observed when the redox moiety is attached to long

flexible linkers such as poly(ethylene glycol) (PEG)22-24 and DNA.25-26 In these

cases, at sufficiently low scan rates the current was proportional to the scan

Chapter 2

8

rate, whereas cycling at increased scan rates showed that the current became

proportional to the square root of the scan rate, following the Randles-Sevcik

equation for diffusing species (equation 2.7):12

𝑖p = 0.4463𝑛𝐹𝐴𝐶 (𝑛𝐹𝜈𝐷

𝑅𝑇)

1

2 (Equation 2.7)

Here, D is the diffusion coefficient and C the concentration. For surface-attached

species, the concentration can be transformed into surface density (Γ) using

Γ=LAC, with L being the layer thickness. From this, information could be

gathered about the dynamics of the flexible linker. For immobilized PEG linkers

it was found that the time response of PEG (Mw of 3400 Da) linkers in a loose

brush conformation, in which the surface-attached polymers are packed in a

sufficiently high density to promote chain stretching, gave similar time

responses as a shorter PEG (Mw of 600 Da) in a mushroom conformation, in

which the individual polymer molecules are isolated from each other. This

indicated that the diffusion coefficients of the longer PEGs were necessarily

higher, which has been attributed to an increased contribution of the spring

constant.23

Similarly, ferrocene moieties that have been linked to single-stranded DNA show

electron transfer that is controlled by diffusion at high scan rates, as could be

determined both experimentally25 and by modelling.27 Upon hybridization of the

single-stranded DNA with its complementary chain, the double-stranded DNA

becomes more rigid which has been shown to affect the diffusional properties

markedly. In this case the regime in which diffusion governs the electron

transfer shifts to lower scan rates, while at higher scan rates the current

decreases drastically, indicating that the rigidity hampers the movement of the

ferrocene to such an extent that it does not reach the surface in the measured

time frame.25 It has been shown that the electron transfer of the double-

stranded DNA in this case occurs via elastic bending of the entire double-

stranded linker (Figure 2.2b).26 It should be noted that this observation applies

only when the DNA is bound to the surface via a short alkane linker (C2 in this

instance), while in contrast, when the linker length is increased to C6 the

electron transfer occurs via the rotational movement around the linker (Figure

2.2c), direct electron transfer through the DNA backbone (Figure 2.2a) could be

excluded.28


9

Figure 2.2: Possible types of electron transport mechanisms in Fc-labeled double-stranded DNA:

(a) Direct electron transfer through the DNA backbone (can be excluded due to the presence of a

diffusion regime). (b) Elastic bending of the double-stranded DNA backbone. (c) Rotational motion

of the DNA rod. Reprinted with permission from Ref. 28. Copyright 2008 American Chemical

Society.

The conformation of molecules on the surface has a marked influence on the

electron transport: studies with ferrocene-tagged PNA on electrodes have

shown that increasing the surface density causes a decrease of the diffusion

constant of two orders of magnitude with an analogous decrease of kET.29

If diffusion plays a role in the electron transfer process, the Nicholson method

can be used for the determination of electron transfer rate constants.30 Using

this method, the peak separation (ΔEp) can be transformed into a dimensionless

parameter ψ, from which the rate constant can be calculated using Equation 2.8.

𝜓 =𝑘ET (

𝐷O

𝐷𝑅)

𝛼

2

√𝜋𝑛𝐹𝜈

𝑅𝑇𝐷O

⁄ (Equation 2.8)

Where DO and DR are the diffusion coefficients of the oxidized and reduced

species, respectively.

If the layer thickness is known, this method can be used for the determination

of rate constants of diffusing species attached to an electrode via a flexible

linker. Rate constants have in this way been determined for electrochemically

active PNA surfaces to study the effect of surface density, chain length and

hybridization. It was shown that with an increase in surface density of a PNA 12-

mer, as shown in Figure 2.3a, the slope of the peak current vs the square root of

the scan rate decreases, indicating a decrease in the diffusion coefficient.

Analogously, the increase in surface density showed a decrease in the electron

Chapter 2

10

transfer rate constants (Figure 2.3b). This was attributed to an increased

distance of the electrochemically active ferrocene to the surface as the layer

becomes more crowded. Similarly, this increase in distance from the surface

resulted in a decrease in rate constants and diffusion coefficients as the linker

length was increased (Figure 2.3c). An increase in kinetics and diffusion was

found upon hybridization with a fully complementary DNA strand due to a

decrease in elasticity of the linker and increased interactions between the

positively biased surface and negatively charged PNA-DNA duplex.29

Figure 2.3: a) Randles Sevcik plot of the immobilization process, a decreased slope of ip vs ν1/2 was

found for increased reaction times (or surface densities, see b) for 12-mer PNA. b) Increase in

surface density (circles) and the decrease in electron transfer kinetics (squares) with increased

reaction time. c) Decrease of the rate constants vs linker length for 3-, 6-, 9-, 12- and 16-mer PNA.

Reprinted with permission from Ref. 29. Copyright 2011 Wiley-VCH Verlag GmbH & Co. KGaA,

Weinheim.

2.2.3 Electrochemical impedance spectroscopy

In electrochemical impedance spectroscopy (EIS), the impedance is measured

by applying a small AC signal over a range of frequencies at a specified

potential.7 The obtained data can be fitted to an equivalent circuit, the easiest

being the Randles circuit, consisting of an element for solution resistance in

series with a parallel combination of the double-layer capacitance (CDL) and an


11

element for the impedance of a faradaic reaction, which for an electrode coated

with an redox-active layer consists of a charge transfer resistance (RCT) and an

element for the adsorption pseudocapacitance (CAD) (Figure 2.4).31-32

The kET can subsequently be calculated using equation 2.9.

𝑘ET =1

2𝑅CT𝐶AD (Equation 2.9)

Impedimetric techniques are often quite time consuming and the system needs

to be in equilibrium, if this is not the case the equivalent circuit needs to be

expanded.7 As a characterization tool it is often used with a redox probe in

solution, for example to characterize the quality of monolayers by way of

assessing the ability to block the diffusing redox probe, which increases the RCT.33

Figure 2.4: Randles equivalent circuit for the impedance of an electrochemically active layer.31-32

Impedimetric sensing techniques based on the change in RCT, or faradaic

impedance, similarly rely on the presence of an electrochemically active

probe.34-35 For example, Barton immobilized antibodies for prostate specific

antigen (PSA, a prostate cancer marker) on an electrode surface. Upon binding

of PSA, the increase in RCT was used as a detection signal.36 In non-faradaic

impedance, no redox probe is necessary as changes in the dielectric properties

on the electrode surface are detected via a change in the capacitance.34, 37 If

upon detection the charge distribution is changed, this will result in a change in

the capacitance. For example, a zwitterionic polymer has been used to

immobilize an anti-insulin antibody on electrode surfaces, and the perturbation

of the double layer caused an associated change in the capacitance

characteristics that provided detection in the fM range in undiluted blood

serum.38

In order to study the kinetics of redox active surfaces, the RCT and CAD need to

be determined (equation 2.8), and the data needs to be fitted to an equivalent

circuit. To overcome this, Bueno has shown that for any redox active surface,

Chapter 2

12

directly plotting the imaginary capacitance vs the frequency (resolved for the

parasitic capacitance, measured at potentials outside of the redox window)

shows a peak at the characteristic frequency which is equal to the rate constant.

The electron transfer rates were determined for azurin films immobilized on

SAMs with increasing thiol layer thicknesses and 11-ferrocene-undecanethiol on

gold electrodes (Figure 2.5a and b, respectively). The electron transfer rates of

azurin films showed a decrease with increased thiol layer thickness. All

determined values were compared with rate constants determined from cyclo-

voltammetric measurements and were shown to be similar.39

Figure 2.5: a) Imaginary capacitance of azurin films on three different supporting thiol layer

thicknesses (hexanethiol, decanethiol and dodecanethiol) after correction for the parasitic

capacitance. The electron transfer rate can be determined directly from the characteristic

frequency. b) Imaginary capacitance of 11-ferrocene-undecanethiol before (red) and after (green)

subtraction of the parasitic capacitance. Reprinted with permission from Ref. 39. Copyright 2012

American Chemical Society.

2.3 Electrochemical DNA sensing

The effect of conformational changes on the electron transfer process has been

exploited to develop E-DNA sensors. The principle of an E-DNA sensor is based

on that of optical molecular beacons40 in which single-stranded DNA backfolds

upon itself at its extremities and forms a stem-loop bringing a fluorophore and

a quencher into close proximity. This loop dissociates upon binding with a

complementary strand. In an E-DNA sensor device the ss-DNA is bound with one

end onto an electrode and via a similar stem-loop brings an electrochemically


13

active ferrocene label into close proximity of the electrode (Figure 2.6). In the

presence of a complementary target, the ferrocene moiety is moved away from

the surface, causing a strongly reduced electron transfer rate. For this system,

target DNA concentrations of 10 pmol were detectable.41

Figure 2.6: A DNA stem-loop bound onto itself at its extremities and attached to an electrode at

one end, bearing a ferrocene tag on the other end. Upon hybridization with its complementary

strand, the ferrocene tag is moved away from the surface resulting in a large change in the redox

current. Reprinted with permission from Ref. 41. Copyright 2003 National Academy of Sciences.

Similar to the previously mentioned ferrocene-modified PNA, which showed a

decrease in kET for increased surface densities, crowding of the surface leads to

an increased signal suppression if the stem-loops are hybridized, because the

densely packed layer prevents collisions of the redox moiety with the electrode

surface.42 The use of a stem-loop was shown not to be necessary for the

detection of complementary DNA strands on a 27-base linear DNA probe. The

binding of a complementary chain changed the rigidity, and with that, the

dynamics of the probe DNA changed sufficiently to give an increased signal

suppression over its stem-loop counterpart. The signal suppression could be

increased even more by increasing the probe density, thereby making the

movement (and subsequent electron transfer) of the hybridized chains more

difficult.43

These and similar E-DNA sensors44-47 based on the stem-loop are ‘signal off’

architectures and provide a decrease in signal upon detection which can give

rise to false positives.41 Even though this can be avoided by the use of

‘multicolor’ redox labels (electrochemically active moieties with a different

Chapter 2

14

E0),48-49 these systems are still limited by a maximum of 100% signal

suppression.43 ‘Signal on’ detection schemes (Figure 2.7) have been developed

based on a variety of schemes, like the pseudoknot,50 triplex DNA interactions,51

inverted stem-loop,52 DNA-PEG-DNA triblock,53 and electrode bound duplex,54

in order to increase the sensitivity, resulting in decreased detection limits

reported as low as 400 fM for the bound duplex.

Figure 2.7: Several schemes for ‘signal on’ detection: (a) the pseudoknot. Reprinted with

permission from Ref. 50. Copyright 2009 American Chemical Society. (b) Triplex DNA interactions.

Reprinted with permission from Ref. 51. Copyright 2014 American Chemical society. (c) inverted

stem-loop. Reprinted with permission from Ref. 52. Copyright 2011 American Chemical Society.

(d) DNA-PEG-DNA triblock. Reprinted with permission from Ref. 53. Copyright 2004 American

Chemical Society. (e) electrode bound duplex. Reprinted with permission from Ref. 54. Copyright

2006 National Academy of Sciences.


15

Aptamer-based electronic sensors employ aptamers, which are short

oligonucleotides selected for their high binding affinities with a wide variety of

biomolecular targets ranging from cells55 to ATP.56 Aptamer-based sensors are

similarly to E-DNA sensors based on the molecular beacon principle, and

detection of an analyte can be based on binding of single-stranded DNA,57 a

displacement of a complementary strand upon binding,58 or interestingly, a

change in the conductivity of the DNA backbone upon binding with an analyte.59

A universal aptamer-based detector has been developed, based on a neutralizer

displacement strategy. The neutralizer is composed of PNA and cationic amino

acids and neutralizes the negative charge of the DNA probe. Due to the presence

of mismatches between the DNA and the neutralizer, the analyte displaces the

neutralizer, causing a significant decrease in the current.60 Figure 2.8 shows ATP

as the analyte, but the method has been shown to work for ATP, cocaine, DNA,

RNA and thrombin, resulting in femto- and attomolar detection limits.

Figure 2.8: Concept of the universal aptamer based detector, binding of the aptamer releases

the neutralizer, thus switching on the electrochemical signal. Reprinted with permission from

Ref. 60. Copyright 2012 Nature Publishing Group.

2.4 Conclusions

A wide array of electrochemical techniques is available and can be used to study

the characteristics of electrochemically active surfaces. A short overview of

several common techniques has been discussed here. Electron transfer rates can

be determined using different techniques like cyclic voltammetry,

chronoamperometry and electrochemical impedance spectroscopy. Next to the

determination of specific electrochemical parameters, the quality of the layers

present on the electrode can be probed. It has been shown that for flexible

linkers like PEG and DNA, the electron transfer is dictated by diffusion of the

Chapter 2

16

probe, which is directly influenced by the flexibility of the linker. As such,

conformational changes of the linker, by changing the probe surface density or

the rigidity upon binding with the complementary DNA strand, can have a

significant effect on the kinetics and diffusion of the probe.

The change in conformation has been exploited for a variety of electrochemical

biosensing methods, like the detection of complementary DNA strands, or other

analytes via binding with DNA aptamers. The resulting change in conformation

affects the signal output in the timeframe of the measurement, resulting in

detection limits sometimes as low as femto-molar. The systems described here

exhibit a relatively easy way of detection and thus provide an attractive

alternative to laborious laboratory tests. Especially in combination with the

increased interest in personalized medicine and point-of-care testing,

electrochemical sensing devices are an attractive candidate for diagnostics.

2.5 References

(1) Adams, D. M.; Brus, L.; Chidsey, C. E. D.; Creager, S.; Creutz, C.; Kagan, C. R.; Kamat, P. V.;

Lieberman, M.; Lindsay, S.; Marcus, R. A.; Metzger, R. M.; Michel-Beyerle, M. E.; Miller, J. R.;

Newton, M. D.; Rolison, D. R.; Sankey, O.; Schanze, K. S.; Yardley, J.; Zhu, X., Charge Transfer on

the Nanoscale: Current Status. J. Phys. Chem. B 2003, 107, 6668-6697.

(2) Labib, M.; Sargent, E. H.; Kelley, S. O., Electrochemical Methods for the Analysis of Clinically

Relevant Biomolecules. Chem. Rev. 2016, 116, 9001-9090.

(3) Grieshaber, D.; MacKenzie, R.; Vörös, J.; Reimhult, E., Electrochemical Biosensors - Sensor

Principles and Architectures. Sensors 2008, 8, 1400-1458.

(4) Plaxco, K. W.; Soh, H. T., Switch-Based Biosensors: A New Approach Towards Real-Time, in Vivo

Molecular Detection. Trends Biotechnol. 2011, 29, 1-5.

(5) Urdea, M.; Penny, L. A.; Olmsted, S. S.; Giovanni, M. Y.; Kaspar, P.; Shepherd, A.; Wilson, P.;

Dahl, C. A.; Buchsbaum, S.; Moeller, G.; Hay Burgess, D. C., Requirements for High Impact

Diagnostics in the Developing World. Nature 2006.

(6) Wang, J., Electrochemical Biosensors: Towards Point-of-Care Cancer Diagnostics. Biosens.

Bioelectron. 2006, 21, 1887-1892.

(7) Eckermann, A. L.; Feld, D. J.; Shaw, J. A.; Meade, T. J., Electrochemistry of Redox-Active Self-

Assembled Monolayers. Coord. Chem. Rev. 2010, 254, 1769-1802.

(8) Yildiz, I.; Raymo, F. M.; Lamberto, M., Self-Assembled Monolayers and Multilayers of

Electroactive Thiols. In Electrochemistry of Functional Supramolecular Systems, John Wiley & Sons,

Inc.: 2010; pp 185-200.

(9) Ricci, F.; Plaxco, K. W., E-DNA Sensors for Convenient, Label-Free Electrochemical Detection of

Hybridization. Microchim. Acta 2008, 163, 149-155.


17

(10) Deng, B.; Lin, Y.; Wang, C.; Li, F.; Wang, Z.; Zhang, H.; Li, X.-F.; Le, X. C., Aptamer Binding Assays

for Proteins: The Thrombin Example—a Review. Anal. Chim. Acta 2014, 837, 1-15.

(11) Jarczewska, M.; Gorski, L.; Malinowska, E., Electrochemical Aptamer-Based Biosensors as

Potential Tools for Clinical Diagnostics. Anal. Methods 2016, 8, 3861-3877.

(12) Bard, A. J.; Faulkner, L. R., Electrochemical Methods: Fundamentals and Applications. 2 ed.;

John Wiley & Sons: New York, 2001.

(13) Chidsey, C. E. D., Free Energy and Temperature Dependence of Electron Transfer at the Metal-

Electrolyte Interface. Science 1991, 251, 919-922.

(14) Laviron, E. E. E., General Expression of the Linear Potential Sweep Voltammogram in the Case

of Diffusionless Electrochemical Systems. J. Electroanal. Chem. Interfacial Electrochem. 1979, 101,

19-28.

(15) Carter, M. T.; Rowe, G. K.; Richardson, J. N.; Tender, L. M.; Terrill, R. H.; Murray, R. W.,

Distance Dependence of the Low-Temperature Electron Transfer Kinetics of (Ferrocenylcarboxy)-

Terminated Alkanethiol Monolayers. J. Am. Chem. Soc. 1995, 117, 2896-2899.

(16) Li, T. T. T.; Weaver, M. J., Intramolecular Electron Transfer at Metal Surfaces. 4. Dependence

of Tunneling Probability Upon Donor-Acceptor Separation Distance. J. Am. Chem. Soc. 1984, 106,

6107-6108.

(17) Ravenscroft, M. S.; Finklea, H. O., Kinetics of Electron Transfer to Attached Redox Centers on

Gold Electrodes in Nonaqueous Electrolytes. J. Phys. Chem. 1994, 98, 3843-3850.

(18) Chidsey, C. E. D.; Bertozzi, C. R.; Putvinski, T. M.; Mujsce, A. M., Coadsorption of Ferrocene-

Terminated and Unsubstituted Alkanethiols on Gold: Electroactive Self-Assembled Monolayers. J.

Am. Chem. Soc. 1990, 112, 4301-4306.

(19) Auletta, T.; van Veggel, F. C. J. M.; Reinhoudt, D. N., Self-Assembled Monolayers on Gold of

Ferrocene-Terminated Thiols and Hydroxyalkanethiols. Langmuir 2002, 18, 1288-1293.

(20) Lee, L. Y. S.; Sutherland, T. C.; Rucareanu, S.; Lennox, R. B., Ferrocenylalkylthiolates as a Probe

of Heterogeneity in Binary Self-Assembled Monolayers on Gold. Langmuir 2006, 22, 4438-4444.

(21) Yoon Suk Lee, L.; Bruce Lennox, R., Ferrocenylalkylthiolate Labeling of Defects in Alkylthiol

Self-Assembled Monolayers on Gold. Phys. Chem. Chem. Phys. 2007, 9, 1013-1020.

(22) Anne, A.; Demaille, C.; Moiroux, J., Elastic Bounded Diffusion. Dynamics of Ferrocene-Labeled

Poly(Ethylene Glycol) Chains Terminally Attached to the Outermost Monolayer of Successively

Self-Assembled Monolayers of Immunoglobulins. J. Am. Chem. Soc. 1999, 121, 10379-10388.

(23) Anne, A.; Moiroux, J., Quantitative Characterization of the Flexibility of Poly(Ethylene Glycol)

Chains Attached to a Glassy Carbon Electrode. Macromolecules 1999, 32, 5829-5835.

(24) Anne, A.; Demaille, C.; Moiroux, J., Terminal Attachment of Polyethylene Glycol (Peg) Chains

to a Gold Electrode Surface. Cyclic Voltammetry Applied to the Quantitative Characterization of

the Flexibility of the Attached Peg Chains and of Their Penetration by Mobile Peg Chains.

Macromolecules 2002, 35, 5578-5586.

Chapter 2

18

(25) Anne, A.; Bouchardon, A.; Moiroux, J., 3′-Ferrocene-Labeled Oligonucleotide Chains End-

Tethered to Gold Electrode Surfaces: Novel Model Systems for Exploring Flexibility of Short DNA

Using Cyclic Voltammetry. J. Am. Chem. Soc. 2003, 125, 1112-1113.

(26) Anne, A.; Demaille, C., Dynamics of Electron Transport by Elastic Bending of Short DNA

Duplexes. Experimental Study and Quantitative Modeling of the Cyclic Voltammetric Behavior of

3′-Ferrocenyl DNA End-Grafted on Gold. J. Am. Chem. Soc. 2006, 128, 542-557.

(27) Huang, K. C.; White, R. J., Random Walk on a Leash: A Simple Single-Molecule Diffusion Model

for Surface-Tethered Redox Molecules with Flexible Linkers. J. Am. Chem. Soc. 2013, 135, 12808-

12817.

(28) Anne, A.; Demaille, C., Electron Transport by Molecular Motion of Redox-DNA Strands:

Unexpectedly Slow Rotational Dynamics of 20-Mer Ds-DNA Chains End-Grafted onto Surfaces Via

C6 Linkers. J. Am. Chem. Soc. 2008, 130, 9812-9823.

(29) Hüsken, N.; Gȩbala, M.; La Mantia, F.; Schuhmann, W.; Metzler-Nolte, N., Mechanistic Studies

of Fc-Pna(·DNA) Surface Dynamics Based on the Kinetics of Electron-Transfer Processes. Chem. -

Eur. J. 2011, 17, 9678-9690.

(30) Nicholson, R. S., Theory and Application of Cyclic Voltammetry for Measurement of Electrode

Reaction Kinetics. Anal. Chem. 1965, 37, 1351-1355.

(31) Randles, J. E. B., Kinetics of Rapid Electrode Reactions. Faraday Discuss. 1947, 1, 11-19.

(32) Creager, S. E.; Wooster, T. T., A New Way of Using Ac Voltammetry to Study Redox Kinetics in

Electroactive Monolayers. Anal. Chem. 1998, 70, 4257-4263.

(33) Beulen, M. W. J.; Bugler, J.; De Jong, M. R.; Lammerink, B.; Huskens, J.; Schönherr, H.; Vancso,

G. J.; Boukamp, B. A.; Wieder, H.; Offenhäuser, A.; Knoll, W.; Van Veggel, F. C. J. M.; Reinhoudt, D.

N., Host-Guest Interactions at Self-Assembled Monolayers of Cyclodextrins on Gold. Chem. - Eur.

J. 2000, 6, 1176-1183.

(34) Santos, A.; Davis, J. J.; Bueno, P. R., Fundamentals and Applications of Impedimetric and

Redox Capacitive Biosensors. J. Anal. Bioanal. Tech. 2014, S7, 15.

(35) Guan, J. G.; Miao, Y. Q.; Zhang, Q. J., Impedimetric Biosensors. J. Biosci. Bioeng. 2004, 97, 219-

226.

(36) Barton, A. C.; Davis, F.; Higson, S. P. J., Labeless Immunosensor Assay for Prostate Specific

Antigen with Picogram Per Milliliter Limits of Detection Based Upon an Ac Impedance Protocol.

Anal. Chem. 2008, 80, 6198-6205.

(37) Berggren, C.; Bjarnason, B.; Johansson, G., Capacitive Biosensors. Electroanalysis 2001, 13,

173-180.

(38) Luo, X.; Xu, M.; Freeman, C.; James, T.; Davis, J. J., Ultrasensitive Label Free Electrical

Detection of Insulin in Neat Blood Serum. Anal. Chem. 2013, 85, 4129-4134.

(39) Bueno, P. R.; Mizzon, G.; Davis, J. J., Capacitance Spectroscopy: A Versatile Approach to

Resolving the Redox Density of States and Kinetics in Redox-Active Self-Assembled Monolayers. J.

Phys. Chem. B 2012, 116, 8822-8829.


19

(40) Bonnet, G.; Tyagi, S.; Libchaber, A.; Kramer, F. R., Thermodynamic Basis of the Enhanced

Specificity of Structured DNA Probes. Proc. Natl. Acad. Sci. U. S. A. 1999, 96, 6171-6176.

(41) Fan, C.; Plaxco, K. W.; Heeger, A. J., Electrochemical Interrogation of Conformational Changes

as a Reagentless Method for the Sequence-Specific Detection of DNA. Proc. Natl. Acad. Sci. U. S.

A. 2003, 100, 9134-9137.

(42) Ricci, F.; Lai, R. Y.; Heeger, A. J.; Plaxco, K. W.; Sumner, J. J., Effect of Molecular Crowding on

the Response of an Electrochemical DNA Sensor. Langmuir 2007, 23, 6827-6834.

(43) Ricci, F.; Lai, R. Y.; Plaxco, K. W., Linear, Redox Modified DNA Probes as Electrochemical DNA

Sensors. Chem. Commun. 2007, 3768-3770.

(44) Lubin, A. A.; Lai, R. Y.; Baker, B. R.; Heeger, A. J.; Plaxco, K. W., Sequence-Specific, Electronic

Detection of Oligonucleotides in Blood, Soil, and Foodstuffs with the Reagentless, Reusable E-DNA

Sensor. Anal. Chem. 2006, 78, 5671-5677.

(45) Lai, R. Y.; Lagally, E. T.; Lee, S.-H.; Soh, H. T.; Plaxco, K. W.; Heeger, A. J., Rapid, Sequence-

Specific Detection of Unpurified Pcr Amplicons Via a Reusable, Electrochemical Sensor. Proc. Natl.

Acad. Sci. U. S. A. 2006, 103, 4017-4021.

(46) Immoos, C. E.; Lee, S. J.; Grinstaff, M. W., Conformationally Gated Electrochemical Gene

Detection. ChemBioChem 2004, 5, 1100-1103.

(47) Mao, Y.; Luo, C.; Ouyang, Q., Studies of Temperature-Dependent Electronic Transduction on

DNA Hairpin Loop Sensor. Nucleic Acids Res. 2003, 31.

(48) Brazill, S. A.; Kim, P. H.; Kuhr, W. G., Capillary Gel Electrophoresis with Sinusoidal

Voltammetric Detection: A Strategy to Allow Four-“Color” DNA Sequencing. Anal. Chem. 2001,

73, 4882-4890.

(49) Hüsken, N.; Gȩbala, M.; Schuhmann, W.; Metzler-Nolte, N., A Single-Electrode, Dual-Potential

Ferrocene-Pna Biosensor for the Detection of DNA. ChemBioChem 2010, 11, 1754-1761.

(50) Cash, K. J.; Heeger, A. J.; Plaxco, K. W.; Xiao, Y., Optimization of a Reusable, DNA Pseudoknot-

Based Electrochemical Sensor for Sequence-Specific DNA Detection in Blood Serum. Anal. Chem.

2009, 81, 656-661.

(51) Idili, A.; Amodio, A.; Vidonis, M.; Feinberg-Somerson, J.; Castronovo, M.; Ricci, F., Folding-

Upon-Binding and Signal-on Electrochemical DNA Sensor with High Affinity and Specificity. Anal.

Chem. 2014, 86, 9013-9019.

(52) Rowe, A. A.; Chuh, K. N.; Lubin, A. A.; Miller, E. A.; Cook, B.; Hollis, D.; Plaxco, K. W.,

Electrochemical Biosensors Employing an Internal Electrode Attachment Site and Achieving

Reversible, High Gain Detection of Specific Nucleic Acid Sequences. Anal. Chem. 2011, 83, 9462-

9466.

(53) Immoos, C. E.; Lee, S. J.; Grinstaff, M. W., DNA-Peg-DNA Triblock Macromolecules for

Reagentless DNA Detection. J. Am. Chem. Soc. 2004, 126, 10814-10815.

Chapter 2

20

(54) Xiao, Y.; Lubin, A. A.; Baker, B. R.; Plaxco, K. W.; Heeger, A. J., Single-Step Electronic Detection

of Femtomolar DNA by Target-Induced Strand Displacement in an Electrode-Bound Duplex. Proc.

Natl. Acad. Sci. U. S. A. 2006, 103, 16677-16680.

(55) Hermann, T.; Patel, D. J., Adaptive Recognition by Nucleic Acid Aptamers. Science 2000, 287,

820-825.

(56) Zuo, X.; Song, S.; Zhang, J.; Pan, D.; Wang, L.; Fan, C., A Target-Responsive Electrochemical

Aptamer Switch (Treas) for Reagentless Detection of Nanomolar Atp. J. Am. Chem. Soc. 2007, 129,

1042-1043.

(57) Radi, A.-E.; Acero Sánchez, J. L.; Baldrich, E.; O'Sullivan, C. K., Reagentless, Reusable,

Ultrasensitive Electrochemical Molecular Beacon Aptasensor. J. Am. Chem. Soc. 2006, 128, 117-

124.

(58) Xiao, Y.; Piorek, B. D.; Plaxco, K. W.; Heeger, A. J., A Reagentless Signal-on Architecture for

Electronic, Aptamer-Based Sensors Via Target-Induced Strand Displacement. J. Am. Chem. Soc.

2005, 127, 17990-17991.

(59) Huang, Y. C.; Ge, B.; Sen, D.; Yu, H.-Z., Immobilized DNA Switches as Electronic Sensors for

Picomolar Detection of Plasma Proteins. J. Am. Chem. Soc. 2008, 130, 8023-8029.

(60) Das, J.; Cederquist, K. B.; Zaragoza, A. A.; Lee, P. E.; Sargent, E. H.; Kelley, S. O., An

Ultrasensitive Universal Detector Based on Neutralizer Displacement. Nat. Chem. 2012, 4, 642-

648.

21

Chapter 3

Self-assembled monolayers on gold of β-cyclodextrin

adsorbates with different anchoring groups

In this chapter, the design of multivalent β-cyclodextrin-based adsorbates

bearing different anchoring groups is described, with the aim to yield stable

monolayers with improved packing and close contact of the cavity to the gold

surface. Towards this end the primary rim of the β-cyclodextrin was decorated

with several functional groups, namely iodide, nitrile, amine, isothiocyanate,

methyl sulfide and isocyanide. Monolayers formed by these adsorbates were

characterized by contact angle measurements, surface plasmon resonance

spectroscopy, polarization-modulation infrared reflection-absorption

spectroscopy, X-ray photoelectron spectroscopy, and electrochemistry. The

nature of the anchoring group influenced the adsorption kinetics, thickness, layer

stability, number of anchoring groups bound to the surface and packing of the

resulting monolayers. Therefore, chemical manipulation of multivalent

adsorbates can be used to modify the properties of their monolayers.

This chapter has been published in: A. Méndez-Ardoy, T. Steentjes, T. Kudernac, J. Huskens,

Langmuir 2014, 30, 3467.

Chapter 3

22

3.1 Introduction

Cyclodextrins (CDs)1 are cyclic oligosaccharides constituted of 6, 7 or 8 (α, β and

γ-CDs respectively) D-glucopyranose units linked by α-(1→4) bonds featuring a

basket-like structure. These have been used extensively as a platform for the

design of adsorbates that assemble to form monolayers with recognition

capabilities. In this sense, they have found a wide range of versatile applications

including oriented immobilization of proteins,2 supramolecular thin film

deposition,3 or as a valuable model for studying multivalent host-guest

interactions.4

Nonetheless the immobilization of the CDs, mostly based on the incorporation

of a varying number of thiol or thioether moieties, it is far from straightforward

and the nature of the anchoring groups and linker have a great impact on the

coverage, orientation and packing and by extrapolation on the host-guest

chemistry due to heterogeneous orientation of the hosts.5-13 While inclusion of

multiple, preferably symmetrically displayed head groups increases the

possibility of achieving orientation of the cyclodextrin core, the strong

multivalent interaction with the substrate hampers lateral mobility and self-

healing.14 The use of multivalently exposed strong gold-binding groups such as

thiols result in a reduced lateral mobility and thus poor packing of the resulting

monolayers.13 Substitution of thiols with long alkyl thioethers with a lower

affinity to gold improved the lateral mobility of the molecules.10 The increased

mobility of the molecules and the interacting additional alkyl chains led to an

increase of surface coverage and an improved order. However the incorporation

of the alkyl chains creates an insulating layer that hampers their use in

applications where electron transfer from or to the electrode is required.

Improving and modulating the adsorption capabilities of multivalent molecules

without hampering the electrical properties remains a challenge. In this sense,

tuning of the affinity of the head group for the substrate might be used for

improving the lateral mobility, although comparative information about the

effect of their chemical nature in the self-assembly properties is in general

scarce.14-17 Furthermore, this concept may take advantage from the fact that

variations of the nature of the head group can change the electron transport

characteristics of the metal-organic interface.18-19

The synthesis of a series of β-CD adsorbates bearing different anchoring groups

and their self-assembly on gold is described here. The anchoring groups were

Self-assembled monolayers on gold of β-cyclodextrin adsorbates

23

selected based on their different affinities for gold and their facile

implementation onto the CD scaffold to ensure the homogeneity of the final

derivatives and eventually the electrode-head group contact. Concretely, β-CD

adsorbates fully functionalized at their primary rim with methyl sulfide,20

isocyanide,21 isothiocyanate,15, 22 nitrile,23 amine24 and iodide25-26 functionalities

were chosen. These adsorbates show lower affinities for gold in comparison to

thiols and provide a convenient set of derivatives well suited for comparative

studies. The kinetics of the self-assembly process and the structure and

electrochemical properties of the monolayer were studied for the presented set

of molecules.

3.2 Results

Figure 3.1 shows the molecular structures of the set of β-CD adsorbates bearing

different anchoring groups on the primary rim of the CD core. Heptaiodo β-CD

adsorbate (1)27 provides the synthetic intermediate for the other derivatives.

Adsorbates 228, 327 and 429 were prepared according to published routes with

some minor modifications.

Figure 3.1: Structure of the β-CD-based adsorbates used in this study.

Chapter 3

24

The synthesis of the per-methylsulfide β-CD 5 was reported previously.9

However the final desired product was obtained only in an inseparable mixture

with incompletely functionalized derivatives. In other to circumvent the

separation problem, an alternative strategy based on the temporary protection

of the secondary rim with acetyl groups was developed (Scheme 3.1a).

Scheme 3.1: Synthesis of adsorbates 5 and 6: i) methyl imidothiocarbamate hydroiodide, Cs2CO3,

DMF, overnight, 44%; ii) MeONa/MeOH, 2.5 h, 82%; iii) formic acid, DCC, Et3N, DCM, 3 h, 88%; iv)

PCl3, DIPA, DCM, 1 h, 63%.

Treatment of compound 7 with methyl imidothiocarbamate hydroiodide in the

presence of Cs2CO3 in N,N-dimethylformamide (DMF) afforded the fully

functionalized derivative 8 after chromatographic purification in 44% yield.

Deacetylation using conventional conditions gave adsorbate 5 in 88% yield after

filtering off the insoluble product. The complete substitution of all seven iodines

was confirmed by proton and carbon NMR showing a single fully symmetric

compound due to its C7 symmetry (see Experimental section, Figure 3.8).

Electrospray ionization mass spectrometry (ESI-MS) and elemental analysis

further supported this conclusion.

The preparation of the per-isocyanide-β-CD 6 was carried out according to the

strategy shown in Scheme 3.1b, consisting of N-formylation followed by

dehydration. The secondary rim was permanently protected as methyl ethers in

order to avoid dehydratation of the hydroxyl groups and to facilitate the

purification procedure. Derivative 9 was treated with a preformed mixture of

formic acid and N,N’-dicyclohexylcarbodiimide (DCC) in dichloromethane (DCM)

to give a mixture of rotamers 10. After dehydration with PCl3 in the presence of


25

an excess of diisopropylamine (DIPA) under careful control of the pH, adsorbate

6 was isolated in a 63% yield. The presence of the isocyano groups was

confirmed by a peak at 159 ppm in the 13C NMR spectrum, as well as an

absorption band at 2148 cm-1 in the infrared attenuated total reflectance (IR-

ATR) spectrum corresponding to stretching of the isocyanide moiety (see

Experimental section, Figures 3.10 and 3.11).

Self-assembled monolayers of adsorbates 2, 4 and 5 were prepared by

immersion of freshly cleaned gold substrates in 0.1 mM adsorbate solutions in

THF-H2O 4:1 for 12-24 h at room temperature, except for adsorbates 1, 3, and

6, which were dissolved in THF-MeOH 4:1, water (pH = 7), and dry degassed

DCM, respectively, for solubility reasons. The resulting monolayers were

characterized by contact angle goniometry, surface plasmon resonance (SPR)

spectroscopy, polarization-modulation infrared reflection-absorption

spectroscopy (PM-IRRAS), X-ray photoelectron spectroscopy (XPS), and

electrochemistry. Table 3.1 summarizes the most relevant SAM properties, all

values are an average of measurements on at least three samples.

Table 3.1: Characterization of the self-assembled monolayers of the β-CD adsorbates on gold.

Adsorbate θa/θr (H2O, deg)a Cml

(F/m2)b

RCT (Ω)c Thickness

(nm)d

1 (I) 45/<20 0.18 82 0.6

2 (CN) 46/<20 0.14 170 1.0

3 (NH2) 38/<20 0.16 63 1.0

4 (NCS) 49/<20 0.14 242 1.1

5 (SMe) 36/<20 0.20 159 0.4

6 (NC) 61/<20 0.12 18 0.5

a Advancing (θa) and receding (θr) contact angles for the monolayer with water.b Capacitance of

the monolayer determined by cyclic voltammetry at a scan rate of 0.1 V s-1 at 0.15 V.c Charge-

transfer resistance determined by electrochemical impedance spectroscopy. d Thickness

determined by SPR.

Advancing contact angles ranged from 36-49o for 1-5, which suggested a

significant hydrophilicity of the substrates. In contrast, the methylated

derivative 6 showed a contact angle of 61o reflecting the more hydrophobic

Chapter 3

26

nature of the methylated secondary rim. Low values for the receding contact

angle further confirm the hydrophilic character of the surfaces. As a control,

bare gold immersed in the same solvent as the β-CD derivatives produced an

advancing contact angle of 66°.

The chemical composition of the monolayers and the presence of the anchoring

groups was confirmed by observing the corresponding vibrational bands in PM-

IRRAS spectra. Figure 3.2a shows the IR-ATR spectrum of neat compound 1 as a

representative example for the whole set of derivatives. The main features of

the spectrum are the strong vibrational bands related to the antisymmetric

glycosidic C-O-C stretching and the coupled C-C/C-O stretching found at 1180-

960 cm-1. Two intense bands are observed in this range. The first band, located

at lower wavenumbers is quasi-degenerate with two components essentially

located at the x, y plane. The other one has an important contribution along the

z axis.30 C-H and O-H bending vibrations are also found in the IR-ATR spectra

between 1412-1160 cm-1, as well as C-H stretching at ca 2900 cm-1 and –OH

stretching (See Experimental section, Figure 3.11). Upon adsorption of the

derivatives, most of the characteristic vibrational bands observed for the neat

compounds are also visible in the PM-IRRAS spectra (Figure 3.2b). Because of

IRRAS selection rules,31 the relative peak intensities between 1190-1010 cm-1

that have different spatial contributions experience changes due to the different

orientation of the CD core. Peaks corresponding to asymmetric and symmetric

C-H stretching are found between 3000 and 2800 cm-1 respectively for 3 and 6.

The presence of a broad band at ca 2079 cm-1 in the monolayer of compound 4

confirms the presence of the isothiocyanate moiety. Although the shape of the

band is changed in the monolayer, no shift was observed in comparison to the

neat compound, which suggests that the vibrational energy does not change

significantly upon adsorption. Monolayers of 6 showed an adsorption band at

2217 cm-1 assignable to the isocyano group, shifted 69 cm-1 to higher

wavenumbers in comparison to the neat compound.


27

Figure 3.2: a) IR-ATR spectra for neat 1. b) PM-IRRAS spectra of monolayers of 1-6.

SPR gave insight into the adsorption kinetics of the molecules and the

monolayer thickness. To a first approximation, at relatively high adsorbate

concentrations to avoid mass transport limitation, the adsorption kinetics

depends on the anchoring group-surface interactions, and thus it can be

regarded as a measure of the binding affinity. Solutions of the adsorbates in

MeOH were employed, except for 3 which was dissolved in water (pH = 7). A

typical process of the formation and the stability of the monolayer can be found

in Figure 3.3a for the adsorption of 6. After recording the baseline with the pure

solvent, the solution of 6 was flowed until the signal leveled off (about 12 min),

indicating that the adsorbate was immobilized on the surface. The sample was

subsequently washed with the pure solvent to investigate the inherent stability

Chapter 3

28

of the monolayer and to remove nonspecifically adsorbed molecules. The final

angle shift indicates the presence of a stable monolayer with an angle shift that

depends on the film thickness and dielectric constant of the adsorbate. The

reflectivity curves of the stabilized monolayers in water were used to obtain an

estimation of the film thickness. The experimental data were fitted to the

theoretical curve generated for a five-layer model (prism/Ti/gold/CD/water)

assuming an εreal = 2.332 for the CD derivatives (Figure 3.3b).

The comparative adsorption profiles and subsequent rinsing steps are shown in

Figure 3.3c. Adsorption of 3 was carried out in water due to solubility issues and

therefore the angle shifts were higher (Figure 3.3d). Due to the slower kinetics

of the adsorption of 1, 2 and 3 to the surface, more concentrated solutions (0.3

mM) were used to achieve maximum adsorption within the same timescale as

when using the 0.1 mM solutions of 4, 5 and 6. The adsorption of 1 did not reach

clear saturation, even after an extended flowing time. The rinsing step for this

compound did not provide reliable information about the removal of the

physisorbed material and therefore is not shown. Instead, the sample was

washed with solvent until a stable signal SPR was observed. The higher shifts

observed for 3, as well as longer washing times needed to achieve stabilization,

suggests that multilayered structures are formed in the process, probably due

to electrostatic interactions with the gold substrate.24 Adsorption curves of 5

saturated quickly in about 40 min with low angle shifts while 4 showed a fast

adsorption with high angle shifts indicating thicker monolayers. We found

thickness values between 0.4 and 1.1 nm assuming that all molecular

monolayers have the same refractive index (Table 3.1). Previous SPR

measurements on densely packed monolayers of thiolated CDs bearing similar

and shorter tethers yielded thicknesses between 0.7 and 1 nm.6,33 These values

correspond well with the upper value 1.1 found for compound 4, that has the

longest anchoring group. The lower thickness values measured for the other

compounds are attributed to the formation of an incomplete monolayer

although variations of the dielectric constant cannot be excluded entirely. We

ruled out multilayer formation as a contribution to the determination of the

thicknesses. As a negative control, the injection of native β-CD (0.3 mM) did not

produce a signal change in the SPR adsorption curve.

The adsorption kinetics of alkylthiols occurs in a two-step process. The first step

is rapid, while the second is slower and is regarded as the rearrangement of

thiols on the surface and the slow adsorption of molecules into newly exposed


29

surface in order to achieve the highest density of packing. Taking both processes

as independent, the film growth can be described by the following expression:

𝑇 = 𝑇1[1 − 𝑒−𝑘1∙𝑡] + 𝑇2[1 − 𝑒−𝑘2∙𝑡] (Equation 3.1)

Where T is the relative thickness, T1 and T2 are limiting thickness of the first and

second step respectively, and k1 and k2 are the corresponding rate constants.33

The adsorption curves were fitted with Equation 3.1, a representative example

is shown in Figure 3.3e. The kinetic data obtained are presented in Table 3.2.

Table 3.2: Rate constants and limiting SPR shift for the adsorption of adsorbates 1-6.

Adsorbate Conc.

(mM)

T1

(nm)

T2

(nm)

k1 × 10-5 (s-1) k2 × 10-5 (s-1) χ2

1 (I) 0.3 0.2 0.6 203 ± 3 14 ± 0.4 1.94

2 (CN) 0.3 0.2 1.1 140 ± 6 14 ± 0.5 0.68

3 (NH2) 0.3 1.6 2.3 383 ± 6 89 ± 1 15.1

4 (NCS) 0.1 0.8 0.4 179 ± 2 17 ± 2 1.43

5 (SMe) 0.1 0.1 0.2 1642 ± 142 91 ± 3 0.26

6 (NC) 0.1 0.4 0.1 3710 ± 130 74 ± 6 0.55

A good fitting is observed except for adsorbate 3, probably due to the

multilayering process, and thus the model might not be well suited to describe

the experimental data. As expected, the second kinetic constant k2 is slower

than k1 by about one order of magnitude which is in agreement with the fast

adsorption, slow reorganization model. In comparison to alkylthiol adsorbates,

the first adsorption kinetics is much slower at comparable concentrations, while

k2 is in the same order of magnitude. The former fact could be interpreted in

basis of the lower affinity of the binding groups used here, as well as the slower

diffusion from the solution to the surface because of the higher molecular

weight of the CD-based adsorbates. On the other hand, with the exception of

adsorbates 4 and 6, the major contribution to the final coverage is given by the

second step as evidenced by the higher T2 values, in contrast to the self-

assembly of thiols. This underlines the importance of the lateral diffusion in the

assembly of these monolayers to allow further binding.

Chapter 3

30

When comparing adsorbates 1-6, while k2 is on the same order of magnitude in

the whole set, k1 is about one order of magnitude higher in 5 and 6.

Figure 3.3: a) SPR adsorption curve corresponding to the adsorption of 6: injection of 0.1 mM

solution in MeOH and washing with MeOH. b) Experimental reflectivity curve (markers) and fit

(solid line) after adsorption of 6 in water. c) Comparative adsorption curves and rinsing steps for

adsorbates 1-5. d) Adsorption curve and rinsing of adsorbate 3 in water. e) Experimental (dashed

lines) and fitted (solid line) adsorption curve of 6.

Electrochemical investigation of the samples provided information about the

relationship between thickness and capacitance of the monolayers and their

permeability towards an external ferro/ferri cyanide redox couple. The

capacitance of SAM-modified electrodes is proportional to the dielectric


31

constant of the separation medium and inversely proportional to the film

thickness, as predicted by the Helmholtz theory.34 Cyclic voltammetry of the

modified gold electrodes in 0.1 M K2SO4 showed no redox reaction in the

measured range 0-0.3 V. Consequently, only non-Faradaic processes contribute

to the current flow at the electrode-solution interface. The values of the

capacitance of the monolayers formed by 1-5 correlate inversely with the

thickness values obtained by SPR (Figure 3.4). The monolayer of 6 produced the

lowest Cml, probably due to a lower dielectric constant of the molecule. The

order of magnitude found for our monolayers is in agreement with the values

found in monolayers of CD adsorbates with short-tethers or cucurbiturils.6, 13, 35

Figure 3.4: Thickness obtained by SPR experiments versus monolayer capacitance obtained by

cyclic voltammetry at 0.15 V at a scan rate of 0.1 V s-1 in 0.1 M K2SO4 in water, the error bars are

the standard deviation determined over measurements on three samples.

Electrochemical impedance spectroscopy (EIS) measurements were carried out

to further study the packing of these monolayers. Nyquist plots (Figure 3.5)

show a semicircle at high frequencies and a straight line at lower frequencies,

indicating difussion controlled charge transport. The charge transfer resistance

values ranged from 18 to 240 Ω, suggesting excellent electron conduction in

comparison to long thioether β-CD monolayers, for which the resistances values

are in kΩ range. 10 This might be related to the packing properties but also to the

inherent presence of unshielded cavities at the surface where the ionic species

can permeate.

The elemental composition of the monolayers and the binding of the adsorbates

was revealed by X-ray photoelectron spectroscopy (XPS) measurements (Table

3.3). For all compounds, a broad band was found for C(1s) at around 286 eV.

Chapter 3

32

Although some carbon contamination was present in some cases, deconvolution

of the carbon signal into three peaks (O-C*-O, C-C*-O and C-C*-C) allowed the

calculation of the elemental ratios with respect to the other elements, as the

majority of the signal of the CD molecules comes from the C-C*-O and O-C*-O

carbons. Signals of the elements constituting the anchoring groups were

observed for all SAMs. The XPS spectrum of a monolayer of per-iodide-β-CD 1

shows the characteristic I(3d5/2) and I(3d3/2) signals. Deconvolution of the I(3d5/2)

signal gave rise to two bands at 619.3 and 621.0 eV with areas of 87% and 13%,

respectively (Figure 3.6a). The lower energy band is assigned to gold-bound

iodide. The higher energy band showed a similar binding energy as unbound

iodide. The nitrogen based adsorbates show the N(1s) nitrogen signal around

399.4 eV (Figure 3.6b). The N(1s) signal of the monolayer based on adsorbate 2

can be fitted with a single band with no apparent shift in comparison to the neat

compound, suggesting that the CN-Au interaction is rather weak.

Table 3.3: XPS binding energies, elemental ratios and fractions of headgroup moieties bound to

gold for SAMs prepared with 1-6

Adsorbate

(X)

Binding energy (eV) C:X ratioa

Exp. (Theor.)

% Head

group

bound

C(1s) N(1s) S(2p3/2) I(3d5/2)

1 (I) 286. 6 619.3 6:1.4 (6:1) 87

2 (CN) 286.3 399.5 7:1.1 (7:1) n. d.

3 (NH2) 286.5 399.4 6:0.9 (6:1) 88

4 (NCS) 286.4 399.7 161.7 7:1.1:0.8

(7:1:1) 61

5 (SMe) 286.5 163.0 7:0.6 (7:1) 100

6 (NC) 286.5 399.7 9:0.8 (9:1) 87


33

Figure 3.5: Representative Nyquist plots for the monolayers prepared by adsorption of 1-6

determined by electrochemical impedance spectroscopy in 1 mM Fe(CN)63-/4- at its formal

potential (0.24 V)

The monolayers of 3 were deconvoluted into two peaks at 399.4 and 401.9 eV

in a ratio 88:12 with the latter assignable to protonated amines.36 This indicates

that the adsorbed molecules interact mostly by strong coordinative interactions

with the gold substrate. The nitrogen signal in the monolayer of isocyanide 6 is

deconvoluted into two bands, the first at 399.7 and the second at 401.5 eV, in a

Chapter 3

34

ratio of 87:13. Since PM-IRRAS did not show unbound isocyanide (Figure 3.2),

we expect that the major signal at 399.7 eV corresponds to bound isocyanide.

The monolayers of 4 and 5 show S(2p) signals centered at 161.7 (broad) and

163.0 eV, respectively (Figure 3.6c). Fitting the S(2p) signal for adsorbate 4 with

two double peaks (for bound and unbound S(2p3/2) and S(2p1/2)) shows that 61%

of the sulfur atoms are bound to gold. The optimal fit of the presence of sulfur

for adsorbate 5 was achieved with two peaks (Figure 3.6c), but due to the low

intensity it can only be considered an estimate. This, together with the energy

shift of 0.2 eV with respect to the neat compound, suggests that all sulfur atoms

of 5 are bound to gold. In all monolayers, a reasonable agreement between the

theoretical and experimental elemental ratios was found for C and N, S, or I,

suggesting that the structural integrity of the molecules is preserved in the self-

assembled monolayers.

Figure 3.6: XPS spectra for (a) I(3d5/2) in monolayers of 1; (b) N(1s) signals for monolayers resulting

from the assembly of 2, 3, 4 and 6; (c) S(2p) signals found for the adsorption of 4 and 5.

3.3 Discussion

There is a lack of data of self-assembled monolayers with different head groups

other than thiols due to the low stability of these monolayers. In our case,


35

multivalent, spatially oriented exposure of the anchoring functionality can be

exploited to enhance affinities10, 35 and increase the adsorption capabilities.

Most of the head groups employed in this study yield rather poorly ordered

and/or incomplete monolayers when presented in monovalent adsorbates, or

even do not exhibit significant adsorption. For instance, amines can be adsorbed

on gold substrates in a vapor-phase environment to give stable monolayers;

however polar solvents such as ethanol disrupt the Au/N interaction because of

its weak nature.37 This was overcome through the multivalent exposure of

amino moieties to achieve stable adsorption, as reported in polyamidoamine

(PAMAM) dendrimers.24, 38-39 Here the β-CD core offers the additional advantage

of directional exposure of the head groups and in that way the maximization of

the interactions does not impose an entropic penalty.

In our comparative studies, we found different kinetics of formation and

structures of monolayers depending on the anchoring group installed. Although

the chemical affinity between the substrate and the head group is probably the

most important contribution to the adsorption kinetics, there might be

additional parameters that influence the overall phenomena, such as the gold-

head group binding geometry. In this sense, the β-CD core can also restrict the

possible reorientation of the head groups and to limit the probability of a

binding event.

A remarkable effect was observed on the adsorption kinetics. In this study,

reorganization provides the more significant contribution for the completion of

the monolayer; for example adsorbate 2 shows slow adsorption kinetics that

could be related to a low head group-substrate affinity. XPS seems to confirm

such observations with no important shift of the N(1s) signal. Therefore,

changing the nature of the head group critically determines the possibility or

reorganization of the adsorbate. As an opposite example, adsorption of 5 is fast

in the first stage, but the reorganization kinetics is not high enough to allow

further adsorption, thus limiting the packing. Longer immersion times do not

promote further packing in this case since the capacitance measurements for

adsorbate 5 suggest lower film thickness, probably meaning sub-monolayer

coverage.

Hydrophilicity of the surface increased upon adsorption of CDs as shown by

water contact angle goniometry. This is expected due to the exposure of the

hydrophilic secondary rim pointing upward the surface and eventually supports

Chapter 3

36

oriented adsorption of the derivatives. PM-IRRAS and XPS further supported the

presence and structural integrity of the adsorbates at the surfaces, since the

most characteristic vibrational bands were observed for the whole set and the

elemental composition was in good agreement with the expected values,

meaning that the constitutive elements are retained at the surface after

monolayer formation. Therefore, cleavage and adsorption of only the anchoring

groups and concomitant release of the β-CD core was excluded. Monolayer

thicknesses from 0.5 to 1.1 nm were comparable to the values reported before

by SPR or AFM for cyclodextrin-based adsorbates.6, 32 Furthermore, SPR and

capacitance measurements were in reasonable agreement. Taking into account

the successful use of multivalent thioethers to yield good quality monolayers

when attached to long alkyl chains,10, 40 adsorbate 5 gave surprisingly the lowest

thickness values. This might indicate the need of interadsorbate interactions to

make this anchoring group effective. Unreliable performance of methyl sulfides

is not unprecedented,15 but the problem seems not to arise from the cleavage

of the thioether on the surface, since the carbon contents found by XPS was still

in good agreement with the theoretical values. EIS provided some information

about packing; the higher packing densities were found for adsorbates 2 and 4.

Interestingly, nitrile-based adsorbates usually gave rise to better quality

monolayers even though a low affinity was observed by SPR. The low values

found for adsorbate 6 might be attributed to a dipole effect rather than poor

packing, since the ν(NC) values obtained are in agreement with >70% saturation

coverages as found in bis and tridentate isocyanides.41

In general, the majority of the anchoring groups of all compounds are used in

the adsorption process, as revealed by XPS and PM-IRRAS. Alkanenitriles may

undergo adsorption through two basic coordination types: η1-type that implies

σ-coordination via the nitrogen atom, or η2-type that involves coordination of

both atoms of the functional group. Clear information about the coordination in

our system is not available since the absence of evident nitrile adsorption bands

on the PM-IRRAS spectra. However, η2-coordination to a variety of metallic

substrates usually produces high shifts on the N(1s) band in XPS spectra (~2.9

eV).42 Fitting of the S(2p) signal for isothiocyanate-based adsorbate 4 evidenced

that 4.3 of 7 isothiocyanato headgroups are involved in the interaction with

gold, and thus isothiocyanates employ the sulfur with the same efficiency as in

long thioether-βCD derivatives and in a more efficient way in comparison to the

per-thio-β-CD.10, 43 The presence of ν(NCS) without evident shift seems to


37

corroborate the presence of unbound head groups. The other sulfur-based

adsorbate 5 indicated 100% of bound sulfur. Finally, PM-IRRAS spectra of

monolayers prepared with isocyanide-based adsorbate 6 exhibited the

characteristic isocyanide stretching band with a shift compatible with a η1

coordination where the isocyanide group is coordinated with on-top gold atoms

through a single lineal carbon-gold bond.41 Here the presence of a single

vibrational band for the isocyanide stretching seems to corroborate the binding

estimation obtained by XPS, with 6 out of 7 anchoring groups bound to the

substrate.

3.4 Conclusions

Modulation of the self-assembly properties of β-CD-based adsorbates was

achieved via homogeneous decoration of the primary rim with an assortment of

functional groups. Methyl sulfide and isocyanide head groups can be

conveniently inserted without compromising the homogeneity of the final

compound through temporal or permanent protection of the secondary rim.

XPS and PM-IRRAS strongly supported the non-destructive attachment to the

surfaces due to the good agreement between the elemental composition found

at the surface and the theoretical value, as well as the presence of the most

characteristic vibrational bands of the β-CD core. The effect of the head group

was evidenced in the adsorption kinetics, amount of material adsorbed and

order of the resulting monolayers. Interestingly, the multivalent exposure of

weakly gold binding anchoring groups such as amines can promote the assembly

in a significant manner and give rise to rather stable monolayers. Good

adsorption capabilities are also achieved with nitriles and isothiocyanates.

Adsorbates 2 and 4 provided monolayers of better quality with higher charge

transfer resistivities and thickness in the monolayer range and constitute the

most promising candidates for further studies in current development in our

laboratories (see also Chapter 4).

3.5 Experimental section

3.5.1 Materials

Reagents and solvents were purchased from commercial sources and used

without further purification unless stated otherwise. 1H and 13C NMR were

recorded at 400 and 100.6 MHz, respectively. 2D COSY and HMQC experiments

were used to assist on NMR peak assignments. Thin-layer chromatography (TLC)

Chapter 3

38

was carried out on aluminum sheets, with visualization by UV light and by

charring with 10% H2SO4. Column chromatography was carried out on silica gel

(230-400 mesh) or basic alumina. ESI-MS spectra were obtained for samples

dissolved in DCM-MeOH, CH3CN-H2O-TFA or THF-MeOH mixtures at low μM

concentrations. DCM and DMF were dried with molecular sieves. Methyl

imidothiocarbamate hydroiodide,44 heptakis(6-deoxy-6-

iodo)cyclomaltoheptaose,27 heptakis (2,3-di-O-acetyl-6-deoxy-6-

iodo)cyclomaltoheptaose,45 heptakis(6-azido-6-deoxy)cyclomaltoheptaose,27

and heptakis(6-deoxy-6-isothiocyanato)cyclomaltoheptaose29 were prepared

according to the known procedures. The analytical data was in a good

agreement with the described data. Heptakis(6-amino-6-

deoxy)cyclomaltoheptaose was prepared following the procedure of Ashton et

al27 and further purified by repeated precipitation of the aqueous solution of

hydrochloride by adding aqueous solution of NH4OH (30%) until reaching the pH

9-10. The precipitate was then filtered off, washed with water, MeOH and

diethyl ether and dried at vacuum.

3.5.2 Synthesis of new adsorbates

Heptakis(6-cyano-6-deoxy)cyclomaltoheptaose (2):

This compound was prepared following the adapted procedure of Baer et al28.

To a solution of heptakis (6-deoxy-6-iodo)cyclomaltoheptaose (1 g, 0.52 mmol)

in dry DMF (17 mL), KCN (310 mg, 4.8 mmol, 1.3 eq) was added and the reaction

mixture was vigorously stirred under Ar atmosphere at 45 oC for 1 day and

subsequently at rt for 1 day. The solution was concentrated to 1/3 of the initial

volume and the residue was poured into 60 mL of cold water and stirred for 30

min. The solid was filtered off and washed with water (3 x 10 mL). The collected

solid was suspended in EtOH (10 mL) and centrifugated (10 min, 7000 rpm) 3

times, then dried at high vacuum to give a white solid. Yield: 354 mg (57%). The

characterization data was according the published data, plus: 1H NMR (400 MHz,

DMSO-d6): δ = 6.05 (bs, 7 H, OH), 5.84 (bs, 7 H, OH), 4.98 (d, 7 H, J1,2 = 3.4 Hz, H-

1), 3.91 (dt, 7 H, H-5), 3.60 (t, 7 H, J2,3 = J3,4 = 9.2 Hz, H-3), 3.40 (m, 14 H, H-2, H-

4), 3.01 (dd, 7 H, J6a,6b = 14.5 Hz, J5,6a = 2.7 Hz, H-6a), 2.90 (dd, 7 H, J5,6b = 8.9 Hz,

H-6b); ESI-MS: m/z = 1199.4 [M + H]+, 1237.8 [M + K]+.

Heptakis(2,3-di-O-acetyl-6-methylthio)cyclomaltoheptaose (8):


39

To a solution of heptakis (2,3-di-O-acetyl-6-deoxy-6-iodo)cyclomaltoheptaose

(1.1 g, 0.44 mmol) and Cs2CO3 (3.6 g, 11 mmol, 3.6 eq.) in dry DMF (39 mL),

methyl imidothiocarbamate hydroiodide (1.4 g, 6.4 mmol, 2.1 eq.) was added

and the reaction mixture was vigorously stirred overnight under Ar atmosphere.

The solution was poured on ice and HCl 0.5 M (200 mL) was added. The aqueous

layer was extracted with DCM (4 x 50 mL). The combined organic layers were

washed with 0.5 M solution of HCl (100 mL) and brine (100 mL), dried (NaSO4),

filtered and concentrated. Traces of DMF were removed under reduced

pressure and the residue was purified by column chromatography using DCM-

MeOH 50:1 → 40:1 as an eluent to give 8 as a white solid. Yield: 370 mg (44%);

Rf = 0.40 (DCM-MeOH 9:1); IR-ATR: 2917, 1741, 1368, 1215, 1025, 728, 601, 466

cm-1; 1H NMR (400 MHz, CDCl3): δ = 5.22 (t, 7 H, H-3), 5.06 (d, 7 H, J1,2 = 3.9 Hz,

H-1), 4.74 (dd, 7 H, J2,3 = 9.8 Hz, H-2), 4.11 (m, 7 H, H-5), 3.73 (t, 7 H, J3,4 = J4,5 =

8.4 Hz, H-4), 2.97 (m, 14 H, H-6a, H-6b), 2.13 (s, 21 H, SCH3), 2.02, 1.99 (2 s, 42

H, 2 COCH3); 13C NMR (100 MHz): δ = 170.9, 169.5 (CO), 96.9 (C-1), 79.1 (C-4),

71.6 (C-5), 71.1 (C-3), 70.8 (C-2), 36.2 (C-6), 20.9 (COCH3), 17.6 (SCH3); ESI-MS:

m/z = 1932.9 [M]+, 1954.9 [M + Na]+; Anal. Calc. for C77H112O42S7∙2 H2O: C, 46.94;

H, 5.93; Found C, 46.97; H, 5.83. NMR spectra are shown in Figure 3.7.

Chapter 3

40

Figure 3.7: 1H (top) and 13C NMR (bottom) spectra (400 and 100.3 MHz, CDCl3) of compound 8.

Heptakis(6-methylthio)cyclomaltoheptaose (5):

To a solution of 8 (370 mg, 0.19 mmol) in MeOH (32 mL), NaOMe 1 M (0.3 mL)

was added and the reaction was stirred at rt. After 30 min a white solid

precipitate appeared and the solution was further vigorously stirred for 2 h. The

solvent was evaporated under reduced pressure and the residue suspended in

water (10 mL), filtrated and further washed with water (3 x 5 mL) and MeOH (5


41

mL), then dried at high vacuum with P2O5 to give a white solid. Yield: 211 mg

(82%); IR-ATR: 3322, 2913, 1038, 585 cm-1; 1H NMR (400 MHz, DMSO-d6): δ =

5.96 (d, 7 H, JH,H = 6.6 Hz, OH), 5.85 (d, 7 H, JH,H = 1.8 Hz, OH) 4.90 (d, 7 H, J1,2 =

3.4 Hz, H-1), 3.78 (m, 7 H, H-5), 3.60 (t, 7 H, J2,3 = J3,4 = 9.6 Hz, H-3), 3.44-3.35 (m,

14 H, H-2, H-4), 3.14 (d, 7 H, J6a,6b = 12.6 Hz, H-6a), 3.60 (dd, 7 H, J5,6b = 8.6 Hz, H-

6b), 2.09 (s, 21 H, SCH3); 13C NMR (100 MHz): δ = 102.1 (C-1), 85.3 (C-4), 72.6 (C-

3), 72.3 (C-2), 71.0 (C-5), 35.1 (C-6), 15.9 (SCH3); ESI-MS: m/z = 1366.9 [M + Na]+;

Anal. Calc. for C49H84O28S7: C, 43.71; H, 6.29. Found C, 43.69; H, 6.41. NMR

spectra are shown in Figure 3.8.


Chapter 3

42

Heptakis(6-deoxy-6-formamido-2,3-di-O-methyl)cyclomaltoheptaose (10):

A solution of DCC (1.16 g, 5.6 mmol, 2.5 eq.) in dry DCM (15 mL) under Ar

atmosphere was cooled down in an ice-bath. 2 M solution of formic acid in dry

DCM (6.7 mL, 13.4 mmol, 6 eq.) was added dropwise to give a white suspension,

and the mixture was further stirred for 15 min. A suspension of heptakis(6-

amino-6-deoxy-2,3-di-O-methyl)cyclomaltoheptaose46-47 (500 mg, 0.32 mmol)

and Et3N (0.31 mL, 2.2 mmol, 1 eq.) in dry DCM (10 mL) was sonicated for 15

min and then added dropwise to the previous solution, and the mixture was

vigorously stirred at 0 oC → rt for 3 h. The suspension was filtered off and the

solid washed with portions of DCM (4 x 5 mL). The organic solvent was washed

with brine (3 x 10 mL) and the organic layer was dried, filtered and concentrated.

The residue was purified by chromatography in DCM-MeOH 2:1 → MeOH to give

a white powder. 10 was isolated as a mixture of rotamers. Yield: 430 mg (88%);

Rf = 0.45 (MeOH); IR-ATR: 3295, 2929, 2832, 1657, 1530, 1382, 1014 cm-1; 1H

NMR (400 MHz, CD3OD): δ = 8.12, 8.09, 8.07, 8.05, 7.97 (5 s, 7 H, CHO), 5.20 (m,

7 H, J1,2 = 3.6 Hz, H-1), 4.20-3.95 (m, 7 H, J6a,6b = 13.8 Hz, J6a,6b = 2.3 Hz, H-6a),

3.88 (m, 7 H, H-5), 3.67 (m, 21 H, OCH3), 3.51 (m, 28 H, H-3, OCH3), 3.45-3.35 (m,

14 H, H-4, H-6b), 3.18 (dd, 7 H, J2,3 = 9.8 Hz, H-2); 13C NMR (100 MHz): δ = 164.2

(CO), 99.9 (C-1), 83.4, 83.2, 82.4 (C-2, C-3, C-4), 71.8 (C-5), 62.0, (OCH3), 54.2

(OCH3), 40.4 (C-6); ESI-MS: m/z = 1542.3 [M + Na]+. NMR spectra are shown in

Figure 3.9.


43


Heptakis(6-deoxy-6-isocyano-2,3-di-O-methyl)cyclomaltoheptaose (6):

An oven dried three-neck flask was cooled down under a steam of dry N2, then

filled with a solution of 10 (150 mg, 0.1 mmol) in dry DCM (5 mL) and DIPA (0.26

mL, 2.6 mmol, 3.8 eq.) under Ar atmosphere. The flask was cooled down in an

ice-bath and PCl3 (0.075 mL, 0.26 mmol, 1.1 eq.) was added dropwise. The

reaction mixture was stirred for 1 h. 0.2 mL of DIPA and 0.025 mL of PCl3 were

added and stirred for 30 min. Then 5% aqueous solution of NaHCO3 was slowly

added (5 mL) and the mixture was stirred for 10 min. DCM was added (30 mL)

and the organic layer was washed with 5% aqueous solution of NaHCO3 (3 x 10

mL) and the organic layer was dried, filtered and concentrated. The residue was

Chapter 3

44

purified by flash column chromatography on dry basic alumina in EtOAc to give

6 as a white solid. The compound was stored at -30 oC under N2. Yield: 86 mg

(63%); Rf = 0.59 (EtOAc); IR-ATR: 2928, 2830, 2148, 1108, 1040, 966, 934, 840

cm-1; 1H NMR (400 MHz, CDCl3): δ = 5.14 (d, 7 H, J1,2 = 3.8 Hz, H-1), 3.87 (m, 7 H,

J6a,5 = 5.3 Hz, H-6a), 3.87 (m, 7 H, H-5), 3.72 (d, 7 H, J6a,6b = 13.8 Hz, H-6b), 3.63

(s, 21 H, OCH3), 3.52 (s, 21 H, OCH3), 3.52-3.47 (m, 14 H, H-3, H-4), 3.21 (dd, 7 H,

J2,3 = 9.2 Hz, H-2); 13C NMR (100 MHz): δ = 159.0 (NC), 98.5 (C-1), 81.5, 81.1, 80.5

(C-2, C-3, C-4), 69.3 (C-5), 61.6 (OCH3), 59.1 (OCH3), 44.1 (C-6); ESI-MS: m/z =

1394.8 [M + H]+, 1416.8 [M + Na]+; Anal. Calc. for C63H91O28N7∙2 H2O: C, 52.90; H,

6.69; N, 6.85. Found C, 52.73; H, 6.38; N, 6.68. NMR spectra are shown in Figure

3.10.



45

Figure 3.11: IR-ATR spectra of compounds 2-6.

3.5.3 Methods

Monolayer preparation:

All glassware used to prepare monolayers was immersed in piranha solution

(conc. H2SO4-H2O2 3:1, warning: piranha should be handled with caution; it can

detonate unexpectedly), washed with copious amount of water and dried in an

oven. Gold substrates were cleaned by an oxygen plasma for 10 min and the

resulting oxide layer was removed by leaving the substrates immersed in EtOH

for 20 min and finally transferred rapidly to 0.1 mM solutions of β-CD adsorbates

in THF-H2O 4:1 (2, 4 and 5), THF-MeOH 4:1 (1), H2O (pH = 7, 3) or DCM (6) for 12-

24 h at room temperature. Isocyanide monolayers were prepared using

degassed DCM under Ar atmosphere. Subsequently, the substrates were

Chapter 3

46

removed from the solution and rinsed with the solvent used in the monolayer

formation, EtOH and dried with a steam of N2.

Monolayer characterization:

Contact angles were measured on a Krüss G10 contact angle setup equipped

with a CCD camera.

Surface Plasmon Resonance Spectroscopy was carried out on glass-supported

gold substrates for SPR (50 nm gold, 2 nm Ti adhesion layer) obtained from

SSENS (Hengelo, The Netherlands). SPR measurements were performed on a

two-channel vibrating mirror angle scan set-up based on the Kretschmann

configuration. 48 The instrument consist of a HeNe laser (JDS Uniphase, 10 mW,

λ = 632.8 nm) whose light passes through a chopper that is connected to a lock-

in amplifier. The light was coupled via a high index prism (LaSFN 9) to an optically

matched glass-supported gold substrate with an index matching oil (Cargille;

series B; nD25 = 1.700 ± 0.002). A Teflon cell was placed on the monolayer via an

O-ring to avoid leakage. SPR experiments were performed in a flow cell system

with Teflon tubing at a continuous flow of 0.15 mL min-1). Solutions of

adsorbates were dissolved in water, MeOH or by adding the minimal amount of

DMSO (2% except in the per-iodide β-CD 1 solution, 10%) and diluted with

MeOH. If DMSO was used, baseline and washing steps were carried out with

MeOH containing the same amount of DMSO. Prior to and after the formation

of the monolayer, reflectivity curves were recorded in MilliQ water after

stabilization of the signal and used to obtain the film thickness. Winspall Version

3.0249 developed by Prof. Dr. W. Knoll’s group was used to simulate and fit

reflectivity curves by generating a 5-layer model system (Prism/Ti/Au/β-CD

film/water), taking the film thickness as fitting parameter and assuming a

dielectric constant of 2.30 for cyclodextrin derivatives.32 The following dielectric

constants were used to simulate the reflectivity curves: LaSFN 9 prism, εreal =

3.42; Ti: εreal = -3.44, εim = 10.2550 and water, εreal = 1.78 The dielectric constants

of gold were obtained by fitting the SPR curve before monolayer formation and

were on average: εreal = -12.80 ± 0.11, εimg = 1.94 ± 0.13.

PM-IRRAS measurements were conducted on a Nicolet FT-IR 6700 and a TOM

optical module (Thermo Scientific) equipped with a Photo Elastic Modulator

(PEM, Hinds Instruments). Spectra were recorded with the p-polarized light

incidence at 82o relative to the surface normal, with the PEM wavenumber set


47

to 1500, 2100 or 2900 cm-1. 200 scans with a resolution of 4 cm-1 at room

temperature were collected in each experiment.

XPS measurements were performed on a Quantera Scanning X-ray Multiprobe

instrument from Physical Electronics, equipped with a monochromatic Al Kα X-

ray source producing approximately 25 W of X-ray power. Spectra were

referenced to the main C 1s peak set at 284.0 eV. A surface area of 1000 μm x

300 μm was scanned with an X-ray beam about 10 μm wide.

All electrochemical measurements were performed on a CH instruments

bipotentiostat 760D. All solutions used during electrochemical measurements

were deaerated with N2 for at least 30 minutes. Cyclic voltammetry

measurements were performed in a three-electrode setup using the SAM-

covered gold plate as the working electrode (area = 0.44 cm2), a platinum disc

as the counter electrode and a Ag/AgCl reference electrode in aqueous 0.1 M

K2SO4. Electrochemical impedance measurements were performed using 2 mm

diameter (CH Instruments) and 1.6 mm diameter (BASi) gold disk electrodes.

Before modification the electrodes were polished using 50 nm alumina particles

(CH Instruments), followed by extensive rinsing with ethanol and 5 minutes of

ultrasonic treatment in ethanol and 5 minutes in MilliQ water. Subsequently the

electrodes were cleaned electrochemically in 0.5 M H2SO4 by applying an

oxidizing potential of 2 V for 5 seconds followed by a reducing potential of -0.35

V for 10 seconds. Then the electrode potential was scanned from -0.25 V to 1.55

V and back for 40 cycles at a scan rate of 100 mV/s. After cleaning the electrodes

were rinsed with MilliQ water and ethanol and dried under a flow of N2 and

placed immediately in the appropriate solution. The measurements were

performed in an aqueous solution of 1 mM Fe(CN)63-/4- in 0.1 M K2SO4 as

supporting electrolyte and measured at the formal potential of the Fe(CN)63-/4-

couple (0.24 V) and an amplitude of 10 mV.

3.6 References

(1) Szejtli, J., Introduction and General Overview of Cyclodextrin Chemistry. Chem. Rev. 1998, 98,

1743-1753.

(2) Yang, L.; Gomez-Casado, A.; Young, J. F.; Nguyen, H. D.; Cabanas-Danés, J.; Huskens, J.;

Brunsveld, L.; Jonkheijm, P., Reversible and Oriented Immobilization of Ferrocene-Modified

Proteins. J. Am. Chem. Soc. 2012, 134, 19199-19206.

Chapter 3

48

(3) Crespo-Biel, O.; Dordi, B.; Reinhoudt, D. N.; Huskens, J., Supramolecular Layer-by-Layer

Assembly: Alternating Adsorptions of Guest- and Host-Functionalized Molecules and Particles

Using Multivalent Supramolecular Interactions. J. Am. Chem. Soc. 2005, 127, 7594-7600.

(4) Gomez-Casado, A.; Dam, H. D.; Yilmaz, D.; Florea, D.; Jonkheijm, P.; Huskens, J., Probing

Multivalent Interactions in a Synthetic Host-Guest Complex by Dynamic Force Spectroscopy. J.

Am. Chem. Soc. 2011, 133, 10849-10857.

(5) Diget, J. S.; Petersen, H. N.; Schaarup-Jensen, H.; Sørensen, A. U.; Nielsen, T. T.; Pedersen, S.

U.; Daasbjerg, K.; Larsen, K. L., Synthesis of β-Cyclodextrin Diazonium Salts and Electrochemical

Immobilization onto Glassy Carbon and Gold Surfaces. Langmuir 2012, 28, 16828-16833.

(6) Perl, A.; Kumprecht, K.; Kraus, T.; Armspach, D.; Matt, D.; Reinhoudt, D. N.; Huskens, J., Self-

Assembled Monolayers of -Cyclodextrin Derivatives on Gold and Their Host-Guest Behavior.

Langmuir 2009, 25, 1534-1539.

(7) McNally, A.; Forster, R. J.; Keyes, T. E., Interfacial Supramolecular Cyclodextrin-Fullerene

Assemblies: Host Reorientation and Guest Stabilization. Phys. Chem. Chem. Phys. 2009, 11, 848-

856.

(8) Kitano, H.; Taira, Y., Inclusion of Bisphenols by a Self-Assembled Monolayer of Thiolated

Cyclodextrin on a Gold Electrode. Langmuir 2002, 18, 5835-5840.

(9) Stine, K. J.; Andraukas, D. M.; Khan, A. R.; Forgo, P.; D'Souza, V. T., Electrochemical Study of

Self-Assembled Monolayers of a β-Cyclodextrin Methyl Sulfide Covalently Linked to

Anthraquinone. J. Electroanal. Chem 1999, 465, 209-218.

(10) Beulen, M. W. J.; Bügler, J.; Lammerink, B.; Geurts, F. A. J.; Biemond, E. M. E. F.; van Leerdam,

K. G. C.; van Veggel, F. C. J. M.; Engbersen, J. F. J.; Reinhoudt, D. N., Self-Assembled Monolayers of

Heptapodant β-Cyclodextrins on Gold. Langmuir 1998, 14, 6424-6429.

(11) Godínez, L. A.; Lin, J.; Muñoz, M.; Coleman, A. W.; Rubin, S.; Parikh, A.; Zawodzinski, T. A. J.;

Loveday, D.; Ferraris, J. P.; Kaifer, A. E., Multilayer Self-Assembly of Amphiphilic Cyclodextrin Hosts

on Bare and Modified Gold Substrates: Controlling Aggregation Via Surface Modification.

Langmuir 1998, 14, 137-144.

(12) Nelles, G.; Weisser, M.; Back, R.; Wohlfart, P.; Wenz, G.; Mittler-Neher, S., Controlled

Orientation of the Cyclodextrin Derivatives Immobilized on Gold Surfaces. J. Am. Chem. Soc. 1996,

118, 5039-5046.

(13) Rojas, M. T.; Königer, R.; Stoddart, J. F.; Kaifer, A. E., Supported Monolayers Containing

Preformed Binding Sites. Synthesis and Interfacial Binding Properties of a Thiolated β-Cyclodextrin

Derivative. J. Am. Chem. Soc. 1995, 117, 336-343.

(14) Chinwangso, P.; Jamison, A. C.; Lee, T. R., Multidentate Adsorbates for Self-Assembled

Monolayer Films. Accounts Chem. Res. 2011, 44, 511-519.

(15) Weidner, T.; Ballav, N.; Zharnikov, M.; Priebe, A.; Long, N. J.; Maurer, J.; Winter, R.;

Rothenberger, A.; Fenske, D.; Rother, D.; Bruh, C.; Fink, H.; Siemeling, U., Dipodal Ferrocene-Based

Adsorbate Molecules for Self-Assembled Monolayers on Gold. Chem. Eur. J. 2008, 14, 4346-4360.


49

(16) Pranger, L.; Goldstein, A.; Tannenbaum, R., Competitive Self-Assembly of Symmetrical,

Difunctional Molecules on Ambient Copper Surfaces. Langmuir 2005, 21, 5396-5404.

(17) Lee, Y.; Morales, G. M.; Yu, L., Self-Assembled Monolayers of Isocyanides on Nickel Electrodes.

Angew. Chem. Int. Ed. 2005, 44, 4228-4231.

(18) Ko, C.-H.; Huang, M.-J.; Fu, M.-D.; Chen, C.-h., Superior Contact for Single-Molecule

Conductance: Electronic Coupling of Thiolate and Isothiocyanate on Pt, Pd, and Au. J. Am. Chem.

Soc. 2010, 132, 756-764.

(19) Heimel, G.; Romaner, L.; Zojer, E.; Brédas, J.-L., Toward Control of the Metal-Organic

Interfacial Electronic Structure in Molecular Electronics: A First-Principles Study on Self-Assembled

Monolayers of -Conjugated Molecules on Noble Metals. Nano Lett. 2007, 7, 932-940.

(20) Troughton, E. B.; Bain, C. D.; Whitesides, G. M., Monolayer Films Prepared by Spontaneous

Self-Assembly of Symmetrical and Unsymmetrical Dialkyl Sulfides from Solution onto Gold

Substrates: Structure, Properties, and Reactivity of Constituent Functional Groups. Langmuir

1988, 4, 365-385.

(21) Angelici, R. J.; Lazar, M., Isocyanide Ligands Adsorbed on Metal Surfaces: Applications in

Catalysis, Nanochemistry, and Molecular Electronics. Inorg. Chem. 2008, 47, 9155-9165.

(22) Kang, H.; Noh, J.; Ganbold, E.-O.; Uuriintuya, D.; Gong, M.-S.; Oh, J. J.; Joo, S.-W., Adsorption

Changes of Cyclohexyl Isothiocyanate on Gold Surfaces. J. Colloid. Interf. Sci. 2009, 336, 648-653.

(23) Steiner, U. B.; Caseri, W. R.; Suter, U. W., Adsorption of Alkanenitriles and Alkanedinitriles on

Gold and Copper. Langmuir 1992, 8, 2771-2777.

(24) Rahman, K. M. A.; Durning, C. J.; Turro, N. J.; Tomalia, D. A., Adsorption of Poly(Amidoamine)

Dendrimers on Gold. Langmuir 2000, 16, 10154-10160.

(25) Yu, Y.; Dubey, M.; Bernasek, S. L.; Dismukes, G. C., Self-Assembled Monolayer of Organic

Iodine on a Au Surface for Attachment of Redox-Active Metal Clusters. Langmuir 2007, 23, 8257-

8263.

(26) Hirayama, M.; Caseri, W. R.; Suter, U. W., Reaction of Long-Chain Iodoalkanes with Gold

Surfaces. J. Colloid Interface Sci. 1998, 202, 167-172.

(27) Ashton, P. R.; Königer, R.; Stoddart, J. F., Amino Acid Derivatives of β-Cyclodextrin. J. Org.

Chem. 1996, 61, 903-908.

(28) Baer, H. H.; Shen, Y.; Santoyo González, F.; Vargas Berenguel, A.; Isac García, J., Synthesis of

a Cycloheptaose Consisting of (1-4)-Linked 7-Amino-6,7-Dideoxy--D-Gluco-Heptopyranosyl

Units: A New Analog of Cyclomaltoheptaose. Carbohydr. Res. 1992, 235, 129-139.

(29) García Fernández, J. M.; Ortiz Mellet, C.; Jiménez Blanco, J. M.; Fuentes Mota, J.; Gadelle, A.;

Coste-Sarguet, A.; Defaye, J., Isothiocyanates and Cyclic Thiocarbamates of ,'-Trehalose,

Sucrose, and Cyclomaltooligosaccharides. Carbohydr. Res. 1995, 268, 57-71.

(30) Mascetti, J.; Castano, S.; Cavagnat, D.; Desbat, B., Organization of β-Cyclodextrin under Pure

Cholesterol, Dmpc, or Dmpg and Mixed Cholesterol/Phospholipid Monolayers. Langmuir 2008, 24,

9616-9622.

Chapter 3

50

(31) Frey, B. L.; Corn, R. M.; Weibel, S. C., Polarization-Modulation Approaches to Reflection-

Absorption Spectroscopy in Handbook of Vibrational Spectroscopy. John Wiley and Sons, Ltd.:

Chichester, UK, 2001; Vol. 2.

(32) Frasconi, M.; Mazzei, F., Electrochemical and Surface Plasmon Resonance Characterization of

β-Cyclodextrin-Based Self-Assembled Monolayers and Evaluation of Their Inclusion Complexes

with Glucocorticoids. Nanotechnology 2009, 20, 285502.

(33) DeBono, R. F.; Loucks, G. D.; Manna, D. D.; Krull, U. J., Self-Assembly of Short and Long-Chain

N-Alkyl Thiols Nto Gold Surfaces: A Real-Time Study Using Surface Plasmon Resonance

Techniques. Can. J. Chem. 1996, 74, 677-688.

(34) Porter, M. D.; Bright, T. B.; Allara, D. L.; Chidsey, C. E. D., Spontaneously Organized Molecular

Assemblies. 4. Structural Characterization of N-Alkyl Thiol Monolayers on Gold by Optical

Ellipsometry, Infrared Spectroscopy, and Electrochemistry. J. Am. Chem. Soc. 1987, 109, 3559-

3568.

(35) An, Q.; Li, G.; Tao, C.; Li, Y.; Wu, Y.; Zhang, W., A General and Efficient Method to Form Self-

Assembled Cucurbit[N]Uril Monolayers on Gold Surfaces. Chem. Commun. 2008, 1989-1991.

(36) Graf, N.; Yegen, E.; Gross, T.; Lippitz, A.; Weigel, W.; Krakert, S.; Terfort, A.; Unger, W. E. S.,

Xps and Nexafs Studies of Aliphatic and Aromatic Amine Species on Functionalized Surfaces. Surf.

Sci. 2009, 603, 2849-2860.

(37) Xu, C.; Sun, L.; Kepley, L. J.; Crooks, R. M., Molecular Interactions between Organized, Surface-

Confined Monolayers and Vapor-Phase Probe Molecules. 6. In-Situ Ftir External Reflectance

Spectroscopy of Monolayer Adsorption and Reaction Chemistry. Anal. Chem. 1993, 65, 2102-2107.

(38) Ujihara, M.; Imae, T., Adsorption Behaviors of Poly(Amido Amine) Dendrimers with an

Azacrown Core and Long Alkyl Chain Spacers on Solid Substrates. J. Colloid. Interf. Sci. 2006, 293,

333-341.

(39) Ito, M.; Toyoko, I.; Aoi, K.; Tsutsumiuchi, K.; Noda, H.; Okada, M., In Situ Investigation of

Adlayer Formation and Adsorption Kinetics of Amphiphilic Surface-Block Dendrimers on Solid

Substrates. Langmuir 2002, 18, 9757-9764.

(40) Schierbaum, K. D.; Weiss, T.; Thoden van Velzen, E. U.; Engbersen, J. F. J.; Reinhoudt, D. N.;

Göpel, W., Molecular Recognition by Self-Assembed Monolayers of Cavitand Receptors. Science

1994, 265, 1413-1415.

(41) Ontko, A. C.; Angelici, R. J., Studies of the Adsorption of Bi- and Tridentate Isocyanides on

Gold Powder. Langmuir 1998, 14, 3071-3078.

(42) Sexton, B. A.; Avery, N. R., Coordination of Acetonitrile (Ch3cn) to Platinum(111): Evidence

for an Η2(C,N) Species. Surf. Sci. 1983, 129, 21-36.

(43) Liu, W.; Zhang, Y.; Gao, X., Interfacial Supramolecular Self-Assembled Monolayers of C60 by

Thiolated β-Cyclodextrin on Gold Surfaces Via Monoanionic C60. J. Am. Chem. Soc. 2007, 129,

4973-4980.


51

(44) Plano, D.; Sanmartín, C.; Moreno, E.; Prior, C.; Calvo, A.; Palop, J. A., Novel Potent

Organoselenium Compounds as Cytotoxic Agents in Prostate Cancer Cells. Bioorg. Med. Chem.

Lett. 2007, 17, 6853-6859.

(45) Karginov, V. A.; Yohannes, A.; Robinson, T. M.; Fahmi, N. E.; Alibek, K.; Hecht, S. M., β-

Cyclodextrin Derivatives That Inhibit Anthrax Lethal Toxin. Bioorg. Med. Chem. 2006, 14, 33-40.

(46) Takeo, K.; Uemura, K.; Mitoh, H., Derivatives of -Cyclodextrin and the Synthesis of 6-O--D-

Glucopyranosyl--Cyclodextrin. J. Carbohydr. Chem. 1988, 7, 293-304.

(47) Boger, J.; Corcoran, R. J.; Lehn, J.-M., Cyclodextrin Chemistry. Selective Modification of All

Primary Hydroxyl Groups of - and β-Cyclodextrins. Helv. Chim. Acta 1978, 61, 2190-2218.

(48) Lenferink, A. T. M.; Kooyman, R. P. H.; Greve, J., An Improved Optical Method for Surface

Plasmon Resonance Experiments. Sens. Actuators B 1991, 3, 261-265.

(49) Knoll, W. Winspall Software V 3.02. http://www.mpip-mainz.mpg.de/groups/knoll/software.

(50) Handbook of Chemistry and Physics. 93rd ed.; Haynes, W. M., Ed. CRC Press: 2013.

Chapter 3

52

53

Chapter 4

Electron transfer rates in host-guest assemblies at

β-cyclodextrin monolayers

The effect of the distance between a β-cyclodextrin (β-CD) host core and a

conductive substrate on the electron-transfer rate of complexed guests as well

as of free-diffusing electrochemically active probes has been studied. First we

have evaluated a set of short-tethered β-CD adsorbates bearing different

anchoring groups in order to get a reliable platform for the study of short-

distance electron transfer. An electrochemically active trivalent guest was

immobilized on these host monolayers in a selective and reversible manner,

providing information about the packing density. Iodine- and nitrile-

functionalized β-CD monolayers gave coverages close to the maximum packing

density. Electron transfer in the presence of Fe(CN)63-/4- studied by impedance

spectroscopy revealed that the electron transfer of the diffusing probe was 3

orders of magnitude faster than when the β-CD cores were separated from the

surface by undecyl chains. When an electrochemically active guest was

immobilized on the surface, electron-transfer rate measurements by cyclic

voltammetry and capacitance spectroscopy showed differences of up to a factor

of 8 for different β-CD monolayers. These results suggest that increasing the

distance between the β-CD core and the underlying conductive substrate leads

to a diminishing of the electron-transfer rate.

This chapter has been published in: A. Méndez-Ardoy, T. Steentjes, B.A. Boukamp, P. Jonkheijm,

T. Kudernac, J. Huskens, Langmuir 2017, in press; doi: 10.1021/acs.langmuir.6b03860.

Chapter 4

54

4.1 Introduction

Electron transfer processes1 are of great importance in a broad range of fields

encompassing biochemistry2-3 and nanoelectronics.4 The understanding of

mechanisms involved in such processes can unveil fundamental information

which is of relevance in, among others, the performance of sensors,5

information storage devices6 or artificial photosynthesis.7-8 Monolayers on solid

substrates offer a versatile platform for the study of such processes because

most of the critical parameters can be tuned by the design of the interfacial

architecture, i.e. length and chemical nature of the bridge.9 Typically, covalent

anchoring of electroactive groups has been employed because homogeneous

monolayers and a high degree of spatial control can be achieved.10 Ferrocene

bound to the surface, normally through alkyl chains, has been broadly used as

an electroactive self-assembled monolayer in order to study and to control

electron transfer processes in a variety of surfaces.11-16 However, covalent

assemblies can only partially model the complex dynamics observed in systems

of high interest, such as biological machinery, where supramolecular

interactions are found ubiquitously. For instance, supramolecular organization

of photosynthetic reaction centers enables electron transfer processes with a

quantum yield close to unity.17

The design of building blocks that can be assembled in a supramolecular fashion

to build up electroactive architectures is a way to mimic biological systems.

Supramolecular complexes have attracted attention as potential constituents of

molecular electronics.18-20 A key point in this sense is the modularity, which

allows structural modifications through non-covalent interactions in a selective,

but reversible manner. This can be exquisitely tuned by the use of

multivalency,21 which has been used for the development of molecular

printboards onto which electroactive dendrimers can be reversibly anchored.22-

25 However, while the distance-dependent electron transfer rate in surface-

confined probes has been broadly addressed in covalently built-up monolayers,

similar effects in host-guest structures have been generally disregarded. This is

especially important if we take into account that when an electrochemically

active guest is recognized, an additional timescale, involving host-guest

complexation, is coupled with the electron transfer process.26-27 Such host-guest

complexation rates have been studied even at the single molecule level.28-30 The

surface architecture could thus become the rate limiting step for a variety of

Electron transfer rates in host-guest assemblies at β-cyclodextrin monolayers

55

processes, i.e. by limiting electron transfer rates.31 Therefore, the design of

platforms, which require a reliable multivalent supramolecular host-guest

assembly and efficient electron transfer, needs a careful evaluation of the

boundaries in which such a limitation may occur.

The aim of the study in this chapter is to evaluate the effect of the distance of

the β-cyclodextrin (β-CD) cavity on the electron transfer rate when the host core

is positioned in close proximity to or distanced from the metallic substrate. For

the latter, we used long alkyl thioether tethers32 as a reference because of their

known good packing capabilities. On the other hand, assembling well-packed

homogeneous β-CD monolayers close to the surface is not trivial because

classical thiol-functionalized β-CDs in general yield limited coverages and

order.33-34 First, we evaluate the packing of several of the β-CD adsorbates

described in Chapter 3. This is critical because the presence of defects on the

structure will influence the electron transfer capabilities. Second, electron

transfer processes are investigated by using diffusing and supramolecularly

anchored electrochemical probes.

4.2 Results and discussion

We have selected an assortment of β-CD-based adsorbates described in Chapter

3. Adsorbates 1-4 feature short tethers on the cyclosaccharidic core and contain

iodine (1, I), isothiocyanate (2, NCS), nitrile (3, CN) and methyl sulfide (4, SMe)

anchoring groups. Adsorbate 5 (C11SC12) is constituted by thioether anchoring

groups embedded in a long alkylic chain (Chart 4.1).

Depending on the anchoring group employed, different layer architectures such

as depicted in Figure 4.1a-c are considered. For short tethers, the varying

affinities to the gold surface will translate into different host coverages on the

surfaces, where low (Figure 4.1a) or high (Figure 4.1b) densities of hosts could

be possible. On the other hand, a high density of adsorbates has been shown for

compound 5 (C11SC12),32 where the β-CD core is separated from the electrode

through alkyl chains (Figure 4.1c). It should be noted that lattice order of the CD

cavities is difficult to prove and that the cartoons depicted in Figure 4.1 are

meant to indicate only differences in density (and concomitantly in the fraction

of exposed bare gold area) and in the distance of the cavity from the surface.

For derivatives 1-4, a detailed analysis described in Chapter 3 has confirmed

multipodal binding, and therefore oriented immobilization, of the host cavities,

albeit with different packing densities and layer quality.35 The packing order of

Chapter 4

56

the monolayer has not been observed for these monolayers. In contrast,

monolayers of 5 have been shown to be well-packed, and a very similar

methylated derivative has been shown by AFM to pack in an ordered fashion.36

Chart 4.1: Chemical structures of β-CD-based adsorbates (1-5) and electrochemical guest probe 6

employed in this study.

Anchoring a ferrocene (Fc) derivative in a supramolecular fashion has a limited

applicability because of a low binding constant to the β-CD cavity (e.g.

ferrocenecarboxylate is complexed by β-CD with a binding constant of Ka = 2.1 ×

103 M-1),37 which in combination with the low solubility of ferrocene derivatives,

allows only partial coverage. To enhance the stability of the supramolecular

complexes on the surface, we prepared trivalent guest 6 (Chart 4.1), which

consists of two strong-binding adamantane moieties linked through

tetra(ethylene glycol) spacers to a benzene ring functionalized with a ferrocene

moiety. It is known that the bis-adamantane motif possesses sufficient length to

bind to two different β-CD hosts (Figure 4.1d),38 therefore allowing divalent

binding on β-CD monolayers at micromolar concentrations, even in the presence

of β-CD in solution. Our evaluation of space-filling models show that the distance

between the adamantane moieties can range from 1.7 nm up to 3.3 nm, while

the ferrocene moiety is less flexible, with the distance to the adamantane

moieties increasing up to 2 nm. Since the diameter of β-CD is ca 1.53 nm, guest

6 could in principle bind trivalently (Figure 4.1e) when the host on the surface is


57

organized in a close-packed structure. Yet monolayers of 5 have been shown to

pack with a periodicity of 2.1 nm,36 which could be sterically problematic for

binding the Fc moiety together with the adamantane moieties.

Figure 4.1: Different monolayers employed in this chapter: a) short tethered, low densely

assembled β-CDs; b) short tethered, high densely assembled β-CDs; c) long tethered, high densely

assembled β-CDs; d) monolayers with divalent binding or (e) monolayers with trivalent binding of

guest 6.

SPR titrations with increasing concentrations of guest 6 in the presence of 1 mM

β-CD were carried out. SPR sensograms showed guest binding that was

reversible after rinsing with aqueous 1 mM β-CD in the absence of the guest (a

titration example is shown in Figure 4.2a), thus regenerating the surface.

Monolayers made from 1 (I), 2 (NCS) and 5 (C11SC12) were studied in this way

(Figure 4.2b). As is evident from the graphs, nonspecific adsorption was

observed for 1 (I) and 2 (NCS) at high guest concentrations, which was also

evidenced by the incomplete removal of the guest by competition with 1 mM

native β-CD. In contrast, guest binding remained fully reversible for adsorbate

5.

Chapter 4

58

Figure 4.2: a) Titration of monolayers of 5 (C11SC12) with increasing concentrations of guest 6 in

the presence of 1 mM β-CD (). In between guest concentration changes, the surface was rinsed

with 1 mM β-CD (Δ). b) SPR titrations with guest 6 in the presence of 1 mM β-CD for monolayers

prepared with adsorbates 1 (I, ), 2 (NCS, ) and 5 (C11SC12, ). Dotted lines represent fittings to

a thermodynamic multivalent model which considers the surface binding of ferrocene and

adamantane moieties in competition with free host on solution.

A thermodynamic model for the binding of the trivalent guest to the surface in

the presence of a competing guest in solution was elaborated on.39 Briefly, this

model contains the affinity constants of β-CD-ferrocene and β-CD-adamantane

guests both in solution (Ki,Fc,l, Ki,Ad,l) and on the surface (Ki,Fc,s, Ki,Ad,s), as well as

two effective concentration terms, accounting for the binding of ferrocene

(Ceff,Fc) and adamantane (Ceff,Ad) on the surface (for more detail, see Experimental

section). The curves in Figure 4.2b very strongly resemble the binding of a

divalent bisadamantyl calixarene to a β-CD monolayer of 5 described before.38

Simulations of SPR sensograms for monolayers constructed with adsorbates 1

(I), 2 (NCS) and 5 (C11SC12) using equal affinities for binding of a guest (Ad or Fc)

moiety in solution or to a surface β-CD site and using effective concentrations

of 0.1 M showed predicted adsorbed masses that were too high when assuming

trivalent binding to the surface. Proper fits were obtained only when assuming

an order of magnitude lower affinity for Ad at the surface or an order of

magnitude lower Ceff for binding of both the Ad and Fc units or when effective

divalent binding was assumed (Ki,Fc,s < 10 M-1 or Ceff,Fc < 1 mM). Of these options,

the first is unlikely because of the way that the Ad group has been attached to

the guest scaffold in comparison to earlier guest molecules used. The second

could indicate a lower packing density, but earlier data on monolayers of 5

always gave Ceff values in the range of 0.1-0.4 M. Therefore, we conclude that


59

the Fc group is not contributing to the overall affinity of the complex with the β-

CD monolayer, making the binding primarily divalent in nature, via both Ad

groups. This is in line with the steric problems of the Fc group noted above. It

does, however, not rule out that part of the Fc groups can show some

interaction with neighboring empty β-CD sites, but only as a minor fraction

(<30%).

Qualitatively, the binding of trivalent 6 to monolayers of adsorbates 1 (I), 2 (NCS)

and 5 (C11SC12) as shown in Figure 4.2 indicates highly comparable binding, both

in affinities and in amounts of adsorbed guests. The different apparent limiting

SPR values could be attributed to differences in packing density, which would

correspond to an order of 2 (NCS) > 5 (C11SC12) > 1 (I), but the higher value for 2

could also come from the higher apparent nonspecific adsorption. Overall, we

conclude that the binding of 6 to the here studied β-CD monolayers is very

similar and essentially divalent. This makes this guest an ideal candidate for

probing the distance variation between the β-CD core and the substrate

imposed by the different β-CD adsorbates and at the same time probing the

effect of a guest, which can only transiently interact with a β-CD cavity, on its

electron-transfer rate to the substrate.

Subsequently, self-assembly of guest 6 was probed electrochemically. Guest 6

was immobilized onto monolayers of 1-5, after which cyclic voltammograms

were measured in the absence of the guest in solution. As a representative

example for short-tethered monolayers, Figure 4.3a shows a cyclic

voltammogram of guest 6 bound to monolayers of 1 (I) which clearly reveals the

presence of both oxidation and reduction peaks of the ferrocene/ferrocenium

couple. The area of both peaks is comparable in a wide range of scan rates (up

to 500 V/s), which indicates that the divalent adamantane anchor prevents the

dissociation of the electroactive moiety from the surface, thus keeping the

electroactive moiety in close proximity. This is in contrast with the observation

of irreversible ferrocene oxidation on β-CD-modified electrodes after the

dissociation from the host cavity.24, 26 After immersion of the guest-

functionalized monolayer in an aqueous solution of 10 mM β-CD, the measured

faradaic signal decreased greatly, suggesting that the binding is reversible upon

competition with a host in solution, which agrees with the SPR experiments

described above. Figure 4.3b shows the data for monolayers of the long-

tethered 5 (C11SC12), which data resemble the observations for 1. The current

increases linearly with the scan rate, as shown for monolayers of 1 in Figure 4.3c,

Chapter 4

60

which confirms that the redox process takes place at the surface of the electrode

and no diffusion of the guest in solution is involved. This was observed for all

monolayers described here. Table 4.1 summarizes the formal apparent

potential, peak separation and full width at half maximum (FWHM) for the

monolayers prepared at room temperature (1, I and 4, SMe), 50 oC (2, NCS and

3, CN) or 60 oC (5, C11SC12), which are the optimized conditions (vide infra).

Figure 4.3. Cyclic voltammograms at 0.1 V/s in 1 M NaClO4, after functionalization with guest 6; a)

monolayers of 1 (I) before (solid line) and after (dashed line) incubating with 10 mM β-CD, and b)

monolayers of adsorbate 5 (C11SC12) before (solid line) and after (dashed line) incubation with 10

mM β-CD. c) Current versus scan rate for monolayers of 1 (I) functionalized with guest 6.

As a general trend, the apparent formal potential E0’ measured in short tethered

hosts is smaller value in comparison with the long tethered adsorbate 5

(C11SC12), and the peak separation at 0.1 V/s is below 59 mV, as expected for

surface-confined species. Full width at half maximum (FWHM) values varied

from 90 to 126 mV. Remarkably, the voltammogram for 5 (C11SC12) shows the

characteristics for an ideal nernstian reaction,40 with a small peak separation (5

mV) and a narrow FWHM (98 mV). The higher values of FWHM found in the case

of guests adsorbed on monolayers of 2 (NCS) might indicate structural disorder

of the host assembly, which results in a broader distribution of the formal

potential.41 Indeed, XPS measurements of monolayers of 2 (NCS) revealed an


61

important fraction of unbound sulfur, and therefore higher levels of disorder in

this monolayer (see Chapter 3).35

Table 4.1: Apparent formal potential, peak separation, FWHM for anodic peak at 0.1 V/s in NaClO4

for monolayers of guest 6 in monolayers prepared with compounds 1-5. Potentials are referenced

versus Ag/AgCl reference electrode.

Adsorbate E0’(mV) Ea-Ec (mV) FWHM

(mV)

1 (I) 496 ± 6 18 ± 2 107 ± 9

2 (NCS) 506 ± 4 55 ± 1 126 ± 12

3 (CN) 525 ± 6 21 ± 6 90 ± 8*

4 (SMe) 482 ± 8 18 ± 5 103 ± 16

5 (C11SC12) 516 ± 6 5 ± 2 98 ± 2

*At room temperature.

We employed compound 6 as an electrochemical probe to quantify the

coverage achieved by the host adsorbate series 1-5. It has been reported that

monolayer formation of macrocyclic, multidentate structures at elevated

temperatures can improve the packing and increase the coverage.42 By

increasing the desorption rate, only adsorbates with higher binding affinities

remain on the surface, while structures showing poor binding affinities tend to

experience desorption, leaving space for new binding events. This has resulted

in an optimal adsorption temperature of 60 oC for adsorbate 5 (C11SC12).36 After

host monolayer formation, the adsorption of guest 6 was carried out at a

concentration of 10 µM guest in the absence of native β-CD by solubilization of

6 in MeOH and dilution in water (the fraction of MeOH v/v is less than 1%), in

order to maximize the fraction of bound hosts. As rationalized above, we

assume the binding to be divalent. We compared coverages for monolayers

prepared at room temperature and at 50 oC by measuring (at room

temperature) the peak areas of ferrocene in cyclic voltammograms at 0.1 V s-1.

The results are shown in Figure 4.4, where we include adsorbate 5 (C11SC12),

adsorbed at 60 oC, as a reference.

Chapter 4

62

Figure 4.4: Comparative coverage of adsorbates 1-5 by using the electrochemical probe 6,

assuming divalent binding of the guest. Different conditions of preparation were employed: gray

bars denote preparation at room temperature, and white bars denote heated samples (50 ºC for

1-4, 60 ºC for 5 (C11SC12)). The thick gray line at 45 pmol cm-2 denotes maximum coverage

determined for adsorbate 5 (C11SC12) based on the experimental lattice constant (2.06 nm).36

Symbol* denotes statistical difference determined by Student´s t-test (p < 0.05). Error margins

indicate standard deviations based measurements of at least 3 independent samples

We observed that the achieved coverage depended both on the nature of the

anchoring group as well as on the preparation procedure. The maximum

coverage expected for adsorbate 5 (C11SC12), based on the experimental lattice

length determined by AFM, is about 45 pmol cm-2.36 Reference compound 5

(C11SC12), assembled at 60 oC, showed high packing densities, even higher (about

30%) than the maximum expected. However, this calculation does not take into

account the surface roughness, which might partially explain this over-

estimation. Coverages of 1-4 assembled at room temperature varied from 23 to

53 pmol cm-2 in comparison to the maximum packing of 5 (C11SC12); these values

range from quasi-higly packed to half-packing coverages. An increase in the

guest coverage on the surface was measured for 2 (NCS) and 3 (CN) when using

an elevated assembly temperature. No significant change was found for 1 (I),

and a decrease in the coverage was observed for 4 (SMe). This confirms that the

good binding properties of 5 (C11SC12) are evidently imposed by the alkyl chains,

since compound 4 (SMe), based also on the thioether motif, desorbs upon

heating, suggesting a rather low binding affinity. Addition structural parameters,

such as heterogeneous orientation of the saccharidic core, could induce a source


63

of error in our quantification of coverage. In Chapter 3, it was described that

adsorbate 2 (NCS) shows about 60% of bound sulfur to the surface,35 which

indicates a disordered structure, combined with a higher film thickness than for

the rest of the adsorbates. This might also suggest that tilted adsorption is

involved; therefore, the binding ability of guest 5 can be strongly influenced by

an irregular host distribution. We therefore conclude that adsorbates 1 (I) and

3 (CN) gave the more reliable short-tethered monolayers.

Electron transfer rates for diffusing and anchored probes. Electrochemical

experiments were carried out at the optimized monolayer preparation

conditions: at increased temperatures for 2 (NCS), 3 (CN) and 5 (C11SC12) and

room temperature for 1 (I) and 4 (SMe). We set out to examine how the

passivation of the surface by the assembly of a host monolayer influences the

electron transfer rate of a freely diffusing electrochemical probe, such as

ferrocene or the [Fe(CN)6]3-/4- redox couple. For ferrocene, the heterogeneous

rate of electron transfer is determined using the Nicholson method,43 for quasi-

irreversible electron transfer dynamics. The peak separation in cyclic

voltammograms can be related to the kinetic parameter Ψ from which k0 can be

calculated using Equation 4.1:

𝛹 = (𝐷𝑂

𝐷𝑅)

𝛼

2 √𝜋𝜈𝐹𝐷𝑂

𝑅𝑇⁄ (Equation 4.1)

Where DO and DR are the diffusion coefficients for the oxidized and reduced

species, respectively, α is the electron-transfer coefficient, F is the Faraday

constant, ν is the scan rate, R is the gas constant and T is the temperature.

When using 126 μM 1,1'-ferrocene-dimethanol (FDM) in aqueous 1 M NaClO4 as

the electrolyte, the peak separations were recorded for scan rates up to 200 V/s

at the various β-CD adsorbate layers from which the kinetic data were extracted

(Table 4.2). The results show that the heterogeneous electron transfer kinetics

for compounds 1-4 are somewhat higher than for compound 5 (C11SC12). This

low difference is attributed to FDM accessing the cavity. On the other hand, the

[Fe(CN)6]3-/4- redox couple cannot diffuse to the electrode through densely

packed monolayers since it is larger than the β-CD core. Cyclic voltammograms

were recorded between 0.1 and 5 V/s and showed significant peak separation

in this region for compounds 1-4, indicative of quasi-irreversible electron

transfer. Adsorbate 1 showed lower values than the others, which is in line with

the better observed packing whereas 2 showed the highest values and a high

Chapter 4

64

deviation indicative of poor (and uncontrolled) packing. The peak separations

recorded for 5 (C11SC12) for these scan rates were too large (ΔEp > 212 mV)

meaning that the electron transfer is irreversible and no kinetic data could be

extracted using this method.

Table 4.2: Comparison of electron transfer rate on monolayers prepared with 1-5.

Adsorbate k0 (cm/s)a k0 (10-3 cm/s)b kET,s (10-3 cm/s)c

1 (I) 0.088 ± 0.013 2.42 ± 1.09 25.4 ± 29.3

2 (NCS) 0.11 ± 0.02 12.4 ± 11.9 19.6 ± 11.7

3 (CN) 0.15 ± 0.02 10.2 ± 6.5 20.8 ± 18.6

4 (SMe) 0.14 ± 0.04 11.9 ± 3.8 100 ± 59.3

5 (C11SC12) 0.062 ± 0.011 - 0.0012 d

a In the presence of FDM, using the Nicholson method. b For the [Fe(CN)6]3-/4- redox couple at 1

mM concentration using the Nicholson method. c For the [Fe(CN)6]3-/4- redox couple at 1 mM

concentration using our previous electrochemical impedance spectroscopy measurements

analyzed as for the determination of kET,s.35 d Calculated from the literature.36

In order to make a better comparison with compound 5 (C11SC12), kET,s values

were calculated from EIS measurements. EIS data was fitted a Randles

equivalent circuit and the kET,s values were obtained from Equation 4.2:44

𝑘𝐸𝑇,𝑠 =𝑅𝑇

𝑛2𝐹2𝐴𝑅𝑐𝑡𝐶 (Equation 4.2)

where R and T are the ideal gas constant and the temperature, n is the number

of electrons transferred, F is the Faraday constant, A the surface area of the

electrode, Rct the electron transfer resistance obtained from the fit, and C the

concentration of [Fe(CN)6]3-/4-. Values for the electron transfer rate obtained in

this way are shown in Table 4.2, the value obtained for 5 (C11SC12) was calculated

from the Rct = 49.2 kΩ.36

These measurements clearly confirmed that the electron transfer rate is

decreased by three orders of magnitude, when the β-CD core is distanced from

the surface through long alkyl chains. Complexing the cavities with an anionic

guest (1-anilinonaphthalene-8-sulfonic acid, 1,8-ANS) gave rise to an increase of

the resistance of the monolayer determined by impedance spectroscopy,45 as


65

shown in Figure 4.5. The magnitude of the increase depended on the coverage

of the host, where clear differences are observed when comparing good (1, I) or

poorly (4, SMe) packed monolayers, which is in agreement with the kET,s trend

described above. Adsorbates 1 (I), 2 (NCS) and 3 (CN) achieved comparable

coverages, which translates in similar electron transfer rates, while adsorbate 4

(SMe), which gives poorly packed monolayers and therefore a higher fraction of

pinholes, showed a higher electron transfer rate.

Figure 4.5: Resistance of the monolayers determined by impedance spectroscopy at increasing

concentrations of 1,8-ANS for monolayers of 1 (I) (black dots) and 4 (SMe) (white boxes).

Experiments were carried out in the presence of 1 mM Fe(CN)63/4- in 0.1 M K2SO4.

We took advantage of the high binding affinity of guest 6 to carry out electron-

transfer measurements of a probe strongly anchored to the surface. In this case,

diffusion from the solution to the surface is not involved. We used cyclic

voltammetry at different scan rates to obtain electron-transfer rates of the

ferrocene moiety, as described by Laviron.46 To minimize the possibility of

having physisorbed material, we immersed monolayers of adsorbates 1-5 in 10

μM solutions of guest 6 in the presence 1 mM of β-CD, and the functionalized

substrates were rinsed and measured in 1 M aqueous NaClO4. Under these

conditions we expect about half the coverage as under the conditions used

above in the absence of β-CD in solution, so approximately half of the β-CD sites

remain unbound. Figure 4.6a shows the peak separation at different scan rates

of guest 6 immobilized on monolayers of 1 (I) and 5 (C11SC12). While increasing

the scan rate, we found higher peak separations for monolayers of 2 (NCS) and

5 (C11SC12), which indicates slower electron-transfer rates. The increase of

anodic and cathodic peak potentials with scan rate is only slightly asymmetric in

Chapter 4

66

most of the range (<200 V/s), which suggest that the kinetics for ferrocene

oxidation and reduction are comparable. For long-tethered adsorbate 5

(C11SC12), at sufficiently high scan rates (> 200 V s-1), the increase of cathodic

peak potential with scan rate was greater than that of the anodic peak potential,

and in addition, the peak corresponding to the reduction of ferrocene broadens.

Figure 4.6: a) Cathodic and anodic currents of guest 6 in monolayers of 1 (I, red circles) and 5

(C11SC12, blue squares) at different scan rates (in V/s) in 1 M NaClO4 (absence of guest in solution).

b) Electron transfer rate of guest 6 obtained by the Laviron method for monolayers prepared with

short tethers: 1 (I), 2 (NCS), 3 (CN), 4 (SMe) and long tethers 5 (C11SC12). Different points for each

adsorbate corresponds to different substrates.

By application of the Laviron formalism, we obtained the electron transfer rates

(kET) and the transfer coefficients. An average transfer coefficient of 0.41 ± 0.07

(short tethered, 1-4) and 0.33 ± 0.11 (5, C11SC12) confirmed a little asymmetry in

the activation energy for the redox process which is more pronounced in long

tethered adsorbates. Electron transfer rates were compared to the coverage of

guest 6, since it is known that differences in coverage might induce differences

in the electron transfer rate due to the different orientation of ferrocene (Figure

4.6b).47 We observed that in general the electron transfer rate for the long-

tethered adsorbate 5 (C11SC12) is in average of 870 s-1 while short-tethered

monolayers 1-4 gave average values of 1124, 715 and 984 s-1 for 1 (I), 2 (NCS)

and 3 (CN) respectively), which is in the range of the values found for ferrocene

anchored to a distance of about 12 methylene units to the surface.48 Adsorbate

4 (SMe) gave higher kET values (1500 s-1), although this measurement was not

highly reliable because of the weakness of the ferrocene signal in this case. In

addition the presence of uncovered host areas might facilitate the electron

transfer. When disregarding adsorbate 2 (NCS), the trend in electron kinetics is


67

similar to the one found for the ferrocyanide couple described above. It is worth

to notice in this respect: i) there is a trend with guest coverage in the kET values

if we compare short-tethered adsorbates 1-4 (since all of the monolayers were

immersed in the same solution, differences in surface coverage of 6 could be

attributed to differences in the packing), ii) this difference is lost in some

substrates for long-tethered 5 (C11SC12), and iii) the magnitude of the difference

is relatively low, 2 (NCS) being the adsorbate showing the lowest electron

transfer rates. However, data obtained by the Laviron method suffer from some

drawbacks, such as nonfaradaic noise or an iR drop. Additionaly, poor definition

of the peaks at higher scan rates makes a reliable analysis difficult.

Table 4.3: Fitted equivalent circuits of impedance data shown in Figure 4.9a-c. Percentages in

parentheses represent the error of the fitting.

At formal potential Outside redox window

Adsorbate RSAM (kΩ) CSAM (µF) RSAM (kΩ) CSAM (µF)

1 (I) 5.9 (9%) 6.8 (0.8%) 14 (28%) 2 (2.5%)

3 (CN) 0.092 (80%) 6 (38%) 5.6 (12%) 3.7 (0.4%)

5 (C11SC12) 0.89 (9.2%) 2.3 (1.6%) 2.3 (232%) 0.9 (22%)

a b

Figure 4.7: Equivalent circuits used in the fitting of impedance spectra at the formal potentials; a)

used for short tethered adsorbates 1 and 3; b) used for long tethered adsorbate 5.

We employed capacitive spectroscopy49 in order to solve the limitations of the

Laviron method. For molecules directly adsorbed to the surface, the imaginary

component C'' of the complex capacitance C*(ω) measured within the redox

potential window contains contributions from the parasitic nonfaradaic and

faradaic charging of the monolayer. The nonfaradaic contribution can be

identified by measuring outside of the redox window and later subtracted to C''.

Chapter 4

68

This corrected C'' function is free from effects from SAM polarization and iR

drops and therefore contains solely information from characteristic frequencies

of the electron transfer of the redox charging49 by assuming that the resistance

of the monolayer does not change importantly with the applied potential.

Fittings of the impedance data taken in and out of the redox window to an

equivalent circuit revealed small changes of the capacitance of the monolayer

(Table 4.3 and Figure 4.7) and might include a source of error. However, these

values as well as the changes in resistance are smaller than observed for the

adsorption of dendrimers.24

As an example, Figure 4.8 shows the transition from non-faradaic (300 mV vs

Ag/AgCl) to mixed faradaic and non-faradaic charging (508 mV vs Ag/AgCl) of

guest 6 complexed on monolayers of 5 (C11SC12) when the potential window is

moved. Figure 4.9a-c shows plots for raw, parasitic and parasitic-corrected C''

for host-guest complexes of 6 in monolayers of 1 (I), 3 (CN) and 5 (C11SC12).

Figure 4.8: Capacitance spectroscopy measurements of guest 6 adsorbed on monolayers of 5

(C11SC12) at different potentials, ranging from outside de redox potential window (300 mV vs

Ag/AgCl) to the redox formal potential (508 mV vs Ag/AgCl).

The observed electron-transfer rates decrease in the order kET in 1 (I) > 3 (CN) >

5 (C11SC12), the latter being up to 6-8 times slower. We notice that these lower

values are comparable to those obtained for monolayers of

ferroceneundecanethiol by the same technique.50 This is an indication of a well-

packed monolayer for 5. In addition, the magnitude of C'' was higher than in

short-tethered adsorbates, probably due to a higher guest coverage. In general,

the ratio between the faradaic and non-faradaic contributions was less than one


69

order of magnitude, which might suggest poor electronic coupling, as observed

for metalloproteins adsorbed on monolayers.50 The differences observed

between the values obtained by the Laviron formalism and the capacitance

method have been attributed to polarization effects that cannot be corrected in

the former.49

Figure 4.9: Electron transfer rates determined by capacitance spectroscopy for the electron

transfer of guest 6 in monolayers of a) 1 (I); b) 3 (CN); c) 5 (C11SC12). Data was collected at the

redox potential (blue lines), outside the redox potential (red lines) and the subtraction gives the

parasitic corrected C' (black line). The electron transfer rate is indicated within the graph. d) kET

measurements for monolayers of 1 (I, red triangles), 3 (CN, white circles) and 5 (C11SC12, blue

squares).

Several reports have shown a decrease of the electron transfer rates of

ferrocene alkyl chains upon increase of the surface coverage. At higher alkyl

ferrocene surface densities, the chains tend to stand up, increasing the distance

between the surface and the ferrocene moieties.51 We varied the surface

coverage in order to compare electron-transfer rates at different surface

densities. Immersion in solutions were performed for guest 6 at 1, 3, 7 and 10

Chapter 4

70

µM for 1 (I) monolayers and 1, 5 and 10 µM for 3 (CN) and 5 (C11SC12). Figure

4.9d shows a set of measurements plotted versus the corresponding ferrocene

coverage.

We observed a trend of increased electron transfer rates at lower guest

coverages, and when coverages are comparable in different adsorbates, the

trend is similar to the mentioned above (kET in 1 (I) > 3 (CN) > 5 (C11SC12)), which

is roughly coherent with our observations using the Laviron method. A possible

explanation is that higher host coverages (reflected in higher guest coverage)

decrease the number of surface defects, therefore reducing the fast electron

transfer through pinholes. This might explain that host samples showing similar

guest coverages can present different electron-transfer rates. Additionally, the

increase in guest packing might require that the Fc moieties orient in a vertical

fashion rather than horizontally, therefore increasing the distance between the

surface and the probe, as reported.51

The small differences observed in the electron transfer rates of adsorbed guest

6 when comparing short and long-tethered adsorbates with both methods

employed is in principle surprising. When a redox moiety is anchored to a

surface separated at a distance d, which is sufficiently short to allow tunneling,

the dependence of the electron-transfer rate kET on d is given by:

kET = kET,0 exp(-βd) (Equation 4.3)

where kET,0 includes the effect of the covalent connection on the electronic

coupling and β is the exponential decay constant, typically about 10 nm-1 for

saturated alkyl chains.52 Therefore the ratio between short-tethered and long-

tethered electron transfer rates (kET,long/kET,short) will be proportional to

exp(β·(dshort – dlong)), which predicts orders of magnitude of difference if dshort is

importantly different from dlong.. Therefore, although distances imposed by the

host adsorbate have an influence on the electron-transfer rate, they do not

provide a complete picture of the charge transport across these host-guest

systems. In this sense, both host and guest coverage as well as other structural

or chemical features of the interfacial layer, for example, the disorder observed

for 2 (NCS) host layers, might play an important role as well. Moreover, as

described above, guest 6 is primarily bound in a divalent fashion, whereby the

Fc moiety is, for most of the time, not close to the host layer. Thus, we can

expect that the divalent binding of 6, with the concomitantly larger distances of

the Fc moiety to the surface possibly mitigated by transient moments of


71

interaction between the Fc moiety and the host cavities, diminishes the relative

contribution of the host layer thickness to the observed differences in electron-

transfer rates.

4.3 Conclusions

We have measured electron transfer rates of probes both in solution and

immobilized on monolayers of β-CD hosts bound directly to the gold surface or

spaced through an alkyl chain layer. For freely diffusing probes, we observed a

decrease in the electron-transfer rate for long-tethered adsorbates of up to 3

orders of magnitude. We have successfully immobilized a trivalent, bis-

Ad/mono-Fc guest on short- and long-tethered β-CD adsorbates by host−guest

interactions. Thermodynamic modeling suggests that the binding of this guest is

predominantly divalent via the stronger-binding Ad moieties. Guest binding is

reversible upon competition with free host in solution, which suggests specific

binding of the multivalent probe to the surface hosts. Electron-transfer rate

measurements revealed faster kinetics in comparison to freely diffusing probes

and a less pronounced difference between short- and long-tethered structures.

While the first could be attributed to a better electronic coupling between the

probe and substrate, the second might be due to the variation in the packing

density (providing the presence of pinholes with facilitated electron-transfer

rates) and the dynamic nature of the assembly, which influences the average

position of the electrochemical probe relative to the surface and thus affects the

transfer rates. This ensemble of results shows that various structural features of

surface host−guest assemblies determine the temporal frame where the

electron transfer takes place. These findings are of importance in the fields of

electronic sensing and molecular electronics because device optimization relies

on strategies to improve the kinetics of the electron transfer, which, to the best

of our knowledge, has not been addressed before in multivalent host−guest

systems. The dynamic nature of host−guest interactions might be an adequate

tool for the future development of architectures with regulated (i.e., externally

switched or induced by conditions) electron-transfer rates by modulating the

affinity of the host−guest binding.


Materials. Reagents and solvents were purchased from commercial sources and

used without further purification unless stated otherwise. 1H and 13C NMR were

recorded at 400 and 100.6 MHz, respectively. 2D COSY and HMQC experiments

Chapter 4

72

were used to assist on NMR peak assignments. Thin-layer chromatography (TLC)

was carried out on aluminum sheets, with visualization by UV light. Column

chromatography was carried out on silica gel (230-400 mesh). ESI-MS spectra

were obtained for samples dissolved in dichloromethane (DCM)-MeOH, at low

μM concentrations. Adsorbates 1-435 and 536 were prepared by reported

procedures.

We additionally developed an alternative procedure for the preparation of 5

based on click thio-ene chemistry: Heptakis6-deoxy-6-[undec-10-

enamido]cyclomaltoheptaose53 was prepared as described in the literature and

further purified by column chromatography in DCM-MeOH 9:1 → 5:1 to give the

heptasubstitued β-CD alkene in 42 % yield. A solution of this derivative (185 mg,

0.08 mmol), AIBN (45 mg, 0.28 mmol, 0.5 eq.) and dodecanethiol (392 mg, 1.9

mmol, 3.5 eq.) in dry, degassed THF (5 mL) was refluxed overnight under argon.

The solvent was evaporated and the residue was dissolved in a little portion of

DCM, then slowly dropped over acetone (50 mL) to give a white precipitate. The

precipitate was filtered off and washed thoroughly with acetone, then dried to

give a white solid (220 mg, 74%). 1H NMR spectra analysis is in agreement with

one reported in the literature (Figure 4.10). Before monolayer preparation, the

product was further purified by column chromatography in DCM-MeOH 20:1 →

9:1. Monolayers showed similar contact angle as the ones prepared following

the reported synthetic procedure.


73

Figure 4.10: 1H NMR spectra (400 MHz, CDCl3) of adsorbate 5.

Synthesis of guest 6: To a solution of the amine precursor54 (194 mg, 0.26 mmol)

in dry DCM (2.5 mL) under Ar, ferrocenoyl chloride (82 mg, 0.33 mmol, 1.3 eq.)

in dry DCM (2.5 mL) and Et3N (45 µL, 0.33 mmol, 1.3 eq.) was added and the

solution was stirred for 2 h. under inert atmosphere. The solvent was

evaporated and the crude was purified twice by chromatography by elution with

EtOAc-petroleum ether 3:1 → 2:1 → EtOAc to give compound 5 as brown syrup.

Yield: 145 mg (57%). Rf = 0.57 (DCM-MeOH 9:1); IR-ATR: 2904, 2851, 1633.5 (CO

amide), 1595, 1534, 1450, 1353, 1293, 1106, 1089, 731cm-1; 1H NMR (400 MHz,

CDCl3): δ = 6.51 (d, 2 H, 4JH,H = 2.2 Hz, Hd), 6.40 (t, 1 H, Hf), 6.06 (t, 1 H, 3JH,H = 5.3

Hz, NH), 4.69 (t, 2 H, JH,H = 1.8 Hz, Fc), 4.47 (d, 2 H, CH2NH), 4.33 (t, 2 H, Fc), 4.18

(s, 5 H, Fc), 4.08 (t, 4 H, 3JH,H = 5.0 Hz, CH2,g), 3.81 (t, 4 H, CH2,h), 3.66-3.53 (m, 24

H, CH2O), 2.12 (bs, 6 H, CHq), 1.73 (d, 12 H, JH,H = 2.7 Hz, CH2,p), 1.59 (m, 12 H,

Chapter 4

74

CH2,r); 13C NMR (100 MHz): δ = 171.2 (C=O), 160.3 (Ce), 107.0 (Cd), 100.6 (Cf),

72.4, 71.4, 71.2, 71.0, 70.7, 69.8, 67.8, 59.3, 43.5 (Cb), 41.6 (Cp), 36.5 (Cr), 30.6

(Cq); ESI-MS: m/z = 972.0 [M]+, 994.0 [M + Na]+; Anal. Calc. for C54H77FeNO11: C,

66.72; H, 7.98; N, 1.44; Found C, 66.52; H, 7.76; N, 1.21. NMR spectra are shown

in Figure 4.11.

Figure 4.11: 1H and 13C NMR spectra (400 and 100.7 MHz, CDCl3) of guest 6.

Monolayers were prepared as described elsewhere.35-36 Gold substrates were

cleaned with piranha, (conc. H2SO4-H2O2 3:1, warning: piranha should be

handled with caution; it can detonate in contact with organic compounds), then

washed with copious amount of water, immersed in EtOH for 20 min and finally

transferred rapidly to 0.1 mM solutions of β-CD adsorbates in THF-H2O 4:1 (2, 3

and 4), THF-MeOH 4:1 (1) or chloroform-EtOH 2:1 (5) for 12 h at room

temperature or 50 oC (1-4) and at 60 oC (5). Subsequently, the substrates were


75

removed from the solution and rinsed with the solvent used in the monolayer

formation, EtOH and dried with a stream of N2. Monolayers of 5 were rinsed

with DCM, EtOH and water at least two times.

Functionalization with guest was carried out in two ways: For host coverage

determination, the guest was dissolved in MeOH and diluted with H2O to give

10 µM solutions (MeOH <1% (v/v)). Substrates were incubated for 10 min with

the guest, then rinsed with H2O, 2 mL β-CD 1 mM, H2O and dried with a stream

of nitrogen. For the rest of the experiments the guest was dissolved in aqueous

β-CD 1 mM solutions with the help of sonication. Substrates were incubated for

10 min with the guest, then rinsed with H2O and dried with a stream of nitrogen.

Competition experiments were carried out by immersion of the substrates in β-

CD 10 mM (twice 15 min).

Surface Plasmon Resonance Spectroscopy was carried out on glass-supported

gold substrates for SPR (50 nm gold, 2 nm Ti adhesion layer) obtained from

SSENS (Hengelo, The Netherlands). SPR measurements were performed on a

two-channel vibrating mirror angle scan set-up based on the Kretschmann

configuration. The instrument consists of a HeNe laser (JDS Uniphase, 10 mW,

λ = 632.8 nm) whose light passes through a chopper that is connected to a lock-

in amplifier. The light was coupled via a high index prism (LaSFN 9) to an optically

matched glass-supported gold substrate with an index matching oil (Cargille;

series B; nD25 = 1.700 ± 0.002). A Teflon cell was placed on the monolayer via an

O-ring to avoid leakage. SPR experiments were performed in a flow cell system

at a continuous flow of 0.15 mL min-1. All the solutions were prepared in the

presence of β-CD 1 mM. Baseline and rinsing steps were carried out with

aqueous β-CD 1 mM.

All electrochemical measurements were performed on a CH instruments

bipotentiostat 760D. All solutions used during electrochemical measurements

were deaerated with N2 for at least 30 minutes. Unless otherwise indicated, all

the measurements were performed in a three-electrode setup using the SAM-

covered gold plate as the working electrode (area = 0.44 cm2), a platinum disc

as the counter electrode and an Ag/AgCl reference electrode in aqueous 1 M

NaClO4. Scan rates used in cyclic voltammetry experiments were 0.1 V s-1 for

coverage determinations and 0.05, 0.1, 0.2, 0.5, 1, 2, 5, 10, 20, 50, 100, 200, 300,

400 and 500 V s-1 for determination of electron transfer rates. All the

measurements were carried out after a first complete scan of preconditioning.

Chapter 4

76

Host coverage quantifications were carried out by determining the charge under

the peak and multiplying by the binding valency determined by the

thermodynamic model. The first 10 scans were averaged for each sample.

Electrochemical impedance measurements for capacitative spectroscopy were

carried out from 1 x 106 - 0.1 Hz and with amplitude of 10 mV at the formal

potential of the measured species after a preconditioning step of 10 s.

Impedance data was converted to capacitance values taking into account Z* =

1/jωC*, and C’’ = φZ’ and C’ = φZ’’, where φ = (ω|Z|2)-1.50 To extract the non-

faradaic contribution to C’’ data was acquired outside the redox potential

between 0.1 - 0.3 V vs Ag/AgCl. Electrochemical impedance measurements were

performed using 2 mm diameter (CH Instruments) and 1.6 mm diameter (BASi)

gold disk electrodes. Before modification the electrodes were polished using 50

nm alumina particles (CH Instruments), followed by extensive rinsing with

ethanol and 5 minutes of ultrasonic treatment in ethanol and 5 minutes in MilliQ

water. Subsequently the electrodes were cleaned electrochemically in 0.5 M

H2SO4 by applying an oxidizing potential of 2 V for 5 seconds followed by a

reducing potential of -0.35 V for 10 seconds. Then the electrode potential was

scanned from -0.25 V to 1.55 V and back for 40 cycles at a scan rate of 100 mV/s.

After cleaning the electrodes were rinsed with MilliQ water and ethanol and

dried under a flow of N2 and placed immediately in the appropriate solution.

Thermodynamic model for the binding to the surface of guest 6. The overall

thermodynamic model for the binding of guest 6 in the presence of competing

native β-CD is showed in Figure 4.12:

Figure 4.12: Overall thermodynamic host-guest equilibrium for adsorption of guest 6 in β-CD

monolayers from a solution containing native β-CD.


77

To simplify the computational cost, first the equilibrium in solution was

considered (Figure 4.13):

Figure 4.13: Thermodynamic host-guest equilibria of guest 6 in a solution of native β-CD.

The total concentration of guest, [Ad-Ad-Fc]tot and total native β-CD on solution

[CD]l,tot are related with the individual species by following mass balance:

[Ad-Ad-Fc]tot = [Ad-Ad-Fc] + [Ad-Ad·CDl-Fc] + [Ad-Ad-Fc·CDl] + [Ad·CDl-Ad·CDl-Fc]

+ [Ad-Ad·CDl-Fc·CDl] + [Ad·CDl-Ad·CDl-Fc·CDl] (1)

[CD]l,tot = [Ad-Ad·CDl-Fc] + [Ad-Ad-Fc·CDl] + 2 ·[Ad·CDl-Ad·CDl-Fc] +

2 [Ad-Ad·CDl-Fc·CDl] + 3 [Ad·CDl-Ad·CDl-Fc·CDl] (2)

Where

[Ad-Ad·CDl-Fc] = 2·Ki,Ad,l [Ad-Ad-Fc][CD]l (3)

[Ad-Ad-Fc·CDl] = Ki,Fc,l [Ad-Ad-Fc][CD]l (4)

[Ad·CDl-Ad·CDl-Fc] = Ki,Ad,l2

[Ad-Ad-Fc][CD]l2 (5)

[Ad-Ad·CDl-Fc·CDl] = 2·Ki,Ad,l Ki,Fc,l [Ad-Ad-Fc][CD]l2 (6)

[Ad·CDl-Ad·CDl-Fc·CDl] = Ki,Ad,l2 Ki,Fc,l [Ad-Ad-Fc][CD]l

3 (7)

Where Ki,Ad,l and Ki,Fc,l denote the thermodynamic host-guest binding constants

for adamantane- native βCD and ferrocene-native βCD in solution for guest 6.

Inserting equations (3-7) into equations (1) and (2) gives:

[Ad-Ad-Fc]tot = [Ad-Ad-Fc] (1 + [CD]l (2Ki, Ad,l + Ki, Fc,l + Ki, Ad,l [CD]l (Ki, Ad,l + 2Ki, Fc,l (1+

½ Ki, Ad,l [CD]l)))) (8)

[CD]l,tot = [CD]l (1+ [Ad-Ad-Fc](2Ki, Ad,l + Ki, Fc,l + 2Ki, Ad,l [CD]l(Ki, Ad,l + 2Ki, Fc,l(1 + 3/4 Ki,

Ad,l [CD]l)))) (9)

We implemented in a spreadsheet equations (3-9) to retrieve the fraction of

bound species (f). Figure 4.14 shows the results of the solution speciation

Chapter 4

78

assuming Ki,Ad,l and Ki,Fc,l equal to 3 × 105 M-1 and 1.2 × 103 M-1, respectively, at

[CD]l,tot = 1 mM.

Figure 4.14. Thermodynamic host-guest equilibrium of guest 6 in a solution of native β-CD

assuming Ki,Ad,l and Ki,Fc,l equal to 3 × 105 M-1 and 1.2 × 103 M-1, respectively.

The total concentration of guest is given by the following expression:

[Ad-Ad-Fc]tot =[Ad-Ad-Fc] + [Ad-Ad-Fc·CDs] + [Ad-Ad·CDl-Fc] + [Ad-Ad-Fc·CDl] +

[Ad-Ad·CDs-Fc] + [Ad-Ad·CDl-Fc·CDs] + [Ad-Ad·CDs-Fc·CDs] + [Ad·CDl-Ad·CDl-Fc] +

[Ad·CDs-Ad·CDs-Fc] + [Ad-Ad·CDl-Fc·CDl] + [Ad-Ad·CDs-Fc·CDl] + [Ad·CDl-Ad·CDs-

Fc] + [Ad·CDl-Ad·CDs-Fc·CDs] + [Ad·CDl-Ad·CDl-Fc·CDs] + [Ad·CDs-Ad·CDs-Fc·CDs] +

[Ad·CDl-Ad·CDl-Fc·CDl] + [Ad·CDs-Ad·CDs-Fc·CDl] + [Ad·CDl-Ad·CDs-Fc·CDl] (10)

Where the concentration of the individual species can be expressed as a

function of [Ad-Ad-Fc], [CD]s and [CD]l :

[Ad-Ad-Fc·CDs] = Ki:Fc,s [Ad-Ad-Fc][CD]s (11)

[Ad-Ad·CDl-Fc] = 2∙Ki:Ad,l [Ad-Ad-Fc][CD]l (12)

[Ad-Ad-Fc·CDl] = Ki:Fc,l [Ad-Ad-Fc][CD]l (13)

[Ad-Ad·CDs-Fc] = 2∙Ki:Ad,s [Ad-Ad-Fc][CD]s (14)

[Ad-Ad·CDl-Fc·CDs] = 2∙Ki:Ad,lKi:Fc,s [Ad-Ad-Fc][CD]s[CD]l (15)

[Ad-Ad·CDs-Fc·CDs] = 2∙Ceff,FcKi:Ad,sKi:Fc,s [Ad-Ad-Fc][CD]s 2/[CD]s,tot (16)

[Ad·CDl-Ad·CDl-Fc] = Ki:Ad,l2[Ad-Ad-Fc][CD]l

2 (17)


79

[Ad-Ad·CDl-Fc·CDl] = 2∙Ki:Fc,lKi:Ad,l [Ad-Ad-Fc][CD]l2 (18)

[Ad·CDl-Ad·CDs-Fc] = Ki:Ad,sKi:Ad,l [Ad-Ad-Fc][CD]l[CD]s (19)

[Ad-Ad·CDs-Fc·CDl] =2∙Ki:Ad,sKi:Fc,l [Ad-Ad-Fc][CD]l[CD]s (20)

[Ad·CDs-Ad·CDs-Fc] = Ceff,AdKi:Ad,s2 [Ad-Ad-Fc][CD]s

2/[CD]s,tot (21)

[Ad·CDl-Ad·CDs-Fc·CDs] = Ki:Ad,lKi:Ad,sKi:Fc,sCeff,Fc [Ad-Ad-Fc][CD]s2[CD]l /[CD]s,tot (22)

[Ad·CDl-Ad·CDl-Fc·CDs] = Ki:Ad,l2Ki:Fc,s [Ad-Ad-Fc][CD]s[CD]l

2 (23)

[Ad·CDs-Ad·CDs-Fc·CDs] = Ceff,AdCeff,FcKi:Ad,s2Ki:Fc,s [Ad-Ad-Fc][CD]s

3/[CD]s2

tot (24)

[Ad·CDl-Ad·CDl-Fc·CDl] = Ki:Fc,lKi:Ad,l2[Ad-Ad-Fc][CD]l

3 (25)

[Ad·CDs-Ad·CDs-Fc·CDl] = Ki:Fc,lKi:Ad,s2 Ceff,Ad[Ad-Ad-Fc][CD]s

2[CD]l /[CD]s,tot (26)

[Ad·CDl-Ad·CDs-Fc·CDl] = Ki:Ad,sKi:Fc,lKi:Ad,l [Ad-Ad-Fc][CD]l2[CD]s (27)

Mass balance of the total βCD on surface [CD]s is given by:

[CD]s,tot = [Ad-Ad-Fc·CDs] + [Ad-Ad·CDs-Fc] + [Ad-Ad·CDl-Fc·CDs] + 2 [Ad-Ad·CDs-

Fc·CDs] + 2 [Ad·CDs-Ad·CDs-Fc] + [Ad-Ad·CDs-Fc·CDl] + [Ad·CDl-Ad·CDs-Fc] + 2

[Ad·CDl-Ad·CDs-Fc·CDs] + [Ad·CDl-Ad·CDl-Fc·CDs] + 3 · [Ad·CDs-Ad·CDs-Fc·CDs] + 2

· [Ad·CDs-Ad·CDs-Fc·CDl] + [Ad·CDl-Ad·CDs-Fc·CDl] (28)

By combining equations (28) and (11-27)

[CD]s =[CD]s,tot/(1+ [Ad-Ad-Fc](Ki:Fc,s + 2 · Ki:Ad,s + [CD]l (2 · Ki:Ad,sKi:Fc,l + Ki:Ad,sKi:Ad,l +

2 · Ki:Ad,lKi:Fc,s +Ki:Ad,l [CD]l (Ki:Fc,s + Ki:Ad,sKi:Fc,l ) + 2 · Ki:Ad,s [CDs] /[CDs]tot (Ki:Ad,lKi:Fc,sCeff,Fc

+ Ki:Fc,lKi:Ad,sCeff,Ad)) + 2 · Ki:Ad,s [CDs]/[CDs]tot (2 · Ceff,FcKi:Fc,s + Ceff,AdKi:Ad,s + 3/2Ceff,FcKi:Fc,s

Ceff,AdKi:Ad,s [Cds]/[CDs]tot))) (29)

Fittings of the model to the experimental data are presented in the main text.

Speciation in the experimental conditions is shown in Figure 4.14.

4.5 References

(1) Launay, J.; Verdaguer, M., Electrons in Molecules: From Basic Principles to Molecular

Electronics. Oxford University Press: 2014.

(2) Moser, C. C.; Keske, J. M.; Warncke, K.; Farid, R. S.; Dutton, P. L., Nature of Biological Electron

Transfer. Nature 1992, 355, 796-802.

(3) Williamson, H. R.; Dow, B. A.; Davidson, V. L., Mechanisms for Control of Biological Electron

Transfer Reactions. Bioorg. Chem. 2014, 57, 213-221.

Chapter 4

80

(4) Adams, D. M.; Brus, L.; Chidsey, C. E. D.; Creager, S.; Creutz, C.; Kagan, C. R.; Kamat, P. V.;

Lieberman, M.; Lindsay, S.; Marcus, R. A.; Metzger, R. M.; Michel-Beyerle, M. E.; Miller, J. R.;

Newton, M. D.; Rolison, D. R.; Sankey, O.; Schanze, K. S.; Yardley, J.; Zhu, X., Charge Transfer on

the Nanoscale: Current Status. J. Phys. Chem. B 2003, 107, 6668-6697.

(5) Waleed Shinwari, M.; Jamal Deen, M.; Starikov, E. B.; Cuniberti, G., Electrical Conductance in

Biological Molecules. Adv. Funct. Mater. 2010, 20, 1865-1883.

(6) Lindsey, J. S.; Bocian, D. F., Molecules for Charge-Based Information Storage. Acc. Chem. Res.

2011, 44, 638-650.

(7) Uosaki, K.; Kondo, T.; Zhang, X.-Q.; Yanagida, M., Very Efficient Visible-Light-Induced Uphill

Electron Transfer at a Self-Assembled Monolayer with a Porphyrin−Ferrocene−Thiol Linked

Molecule. J. Am. Chem. Soc. 1997, 119, 8367-8368.

(8) Yamada, H.; Imahori, H.; Nishimura, Y.; Yamazaki, I.; Ahn, T. K.; Kim, S. K.; Kim, D.; Fukuzumi,

S., Photovoltaic Properties of Self-Assembled Monolayers of Porphyrins and Porphyrin−Fullerene

Dyads on Ito and Gold Surfaces. J. Am. Chem. Soc. 2003, 125, 9129-9139.

(9) Love, J. C.; Estroff, L. A.; Kriebel, J. K.; Nuzzo, R. G.; Whitesides, G. M., Self-Assembled

Monolayers of Thiolates on Metals as a Form of Nanotechnology. Chem. Rev. 2005, 105, 1103-

1170.

(10) Eckermann, A. L.; Feld, D. J.; Shaw, J. A.; Meade, T. J., Electrochemistry of Redox-Active Self-

Assembled Monolayers. Coord. Chem. Rev. 2010, 254, 1769-1802.

(11) Ruther, R. E.; Cui, Q.; Hamers, R. J., Conformational Disorder Enhances Electron Transfer

through Alkyl Monolayers: Ferrocene on Conductive Diamond. J. Am. Chem. Soc. 2013, 135, 5751-

5761.

(12) Liu, B.; Bard, A. J.; Mirkin, M. V.; Creager, S. E., Electron Transfer at Self-Assembled

Monolayers Measured by Scanning Electrochemical Microscopy. J. Am. Chem. Soc. 2004, 126,

1485-1492.

(13) Smalley, J. F.; Finklea, H. O.; Chidsey, C. E. D.; Linford, M. R.; Creager, S. E.; Ferraris, J. P.;

Chalfant, K.; Zawodzinsk, T.; Feldberg, S. W.; Newton, M. D., Heterogeneous Electron-Transfer

Kinetics for Ruthenium and Ferrocene Redox Moieties through Alkanethiol Monolayers on Gold.

J. Am. Chem. Soc. 2003, 125, 2004-2013.

(14) Napper, A. M.; Liu, H.; Waldeck, D. H., The Nature of Electronic Coupling between Ferrocene

and Gold through Alkanethiolate Monolayers on Electrodes: The Importance of Chain

Composition, Interchain Coupling, and Quantum Interference. J. Phys. Chem. B 2001, 105, 7699-

7707.



(16) Chidsey, C. E. D.; Bertozzi, C. R.; Putvinski, T. M.; Mujsce, A. M., Coadsorption of Ferrocene-

Terminated and Unsubstituted Alkanethiols on Gold: Electroactive Self-Assembled Monolayers. J.

Am. Chem. Soc. 1990, 112, 4301-4306.


81

(17) Allen, J. P.; Williams, J. C., Photosynthetic Reaction Centers. FEBS Lett. 1998, 438, 5-9.

(18) Fujii, S.; Tada, T.; Komoto, Y.; Osuga, T.; Murase, T.; Fujita, M.; Kiguchi, M., Rectifying Electron-

Transport Properties through Stacks of Aromatic Molecules Inserted into a Self-Assembled Cage.

J. Am. Chem. Soc. 2015, 137, 5939-5947.

(19) Li, F.; Ito, T., Complexation-Induced Control of Electron Propagation Based on Bounded

Diffusion through Nanopore-Tethered Ferrocenes. J. Am. Chem. Soc. 2013, 135, 16260-16263.

(20) Wimbush, K. S.; Reus, W. F.; van der Wiel, W. G.; Reinhoudt, D. N.; Whitesides, G. M.; Nijhuis,

C. A.; Velders, A. H., Control over Rectification in Supramolecular Tunneling Junctions. Angew.

Chem. Int. Ed. 2010, 49, 10176-10180.

(21) Huskens, J., Multivalent Interactions at Interfaces. Curr. Opin. Chem. Biol. 2006, 10, 537-543.

(22) Nijhuis, C. A.; Huskens, J.; Reinhoudt, D. N., Binding Control and Stoichiometry of Ferrocenyl

Dendrimers at a Molecular Printboard. J. Am. Chem. Soc. 2004, 126, 12266-12267.

(23) Nijhuis, C. A.; Yu, F.; Knoll, W.; Huskens, J.; Reinhoudt, D. N., Multivalent Dendrimers at

Molecular Printboards: Influence of Dendrimer Structure on Binding Strength and Stoichiometry

and Their Electrochemically Induced Desorption. Langmuir 2005, 21, 7866-7876.

(24) Nijhuis, C. A.; Boukamp, B. A.; Ravoo, B. J.; Huskens, J.; Reinhoudt, D. N., Electrochemistry of

Ferrocenyl Dendrimer−β-Cyclodextrin Assemblies at the Interface of an Aqueous Solution and a

Molecular Printboard. J. Phys. Chem. C 2007, 111, 9799-9810.

(25) Yang, L.; Gomez-Casado, A.; Young, J. F.; Nguyen, H. D.; Cabanas-Danés, J.; Huskens, J.;

Brunsveld, L.; Jonkheijm, P., Reversible and Oriented Immobilization of Ferrocene-Modified

Proteins. J. Am. Chem. Soc. 2012, 134, 19199-19206.

(26) Mallon, C. T.; Forster, R. J.; Keyes, T. E., Mechanism and Release Rates of Surface Confined

Cyclodextrin Guests. Analyst 2011, 136, 5051-5057.

(27) Mallon, C. T.; McNally, A.; Keyes, T. E.; Forster, R. J., Mercury−Platinum Tunneling Junctions

Incorporating Supramolecular Host−Guest Assemblies. J. Am. Chem. Soc. 2008, 130, 10002-10007.

(28) Schönherr, H.; Beulen, M. W. J.; Bügler, J.; Huskens, J.; van Veggel, F. C. J. M.; Reinhoudt, D.

N.; Vancso, G. J., Individual Supramolecular Host−Guest Interactions Studied by Dynamic Single

Molecule Force Spectroscopy. J. Am. Chem. Soc. 2000, 122, 4963-4967.

(29) Zapotoczny, S.; Auletta, T.; de Jong, M. R.; Schönherr, H.; Huskens, J.; van Veggel, F. C. J. M.;

Reinhoudt, D. N.; Vancso, G. J., Chain Length and Concentration Dependence of Β-

Cyclodextrin−Ferrocene Host−Guest Complex Rupture Forces Probed by Dynamic Force

Spectroscopy. Langmuir 2002, 18, 6988-6994.

(30) Auletta, T.; de Jong, M. R.; Mulder, A.; van Veggel, F. C. J. M.; Huskens, J.; Reinhoudt, D. N.;

Zou, S.; Zapotoczny, S.; Schönherr, H.; Vancso, G. J.; Kuipers, L., β-Cyclodextrin Host−Guest

Complexes Probed under Thermodynamic Equilibrium: Thermodynamics and Afm Force

Spectroscopy. J. Am. Chem. Soc. 2004, 126, 1577-1584.

(31) Slinker, J. D.; Muren, N. B.; Renfrew, S. E.; Barton, J. K., DNA Charge Transport over 34 nm.

Nat Chem 2011, 3, 228-233.

Chapter 4

82

(32) Beulen, M. W. J.; Bügler, J.; Lammerink, B.; Geurts, F. A. J.; Biemond, E. M. E. F.; van Leerdam,

K. G. C.; van Veggel, F. C. J. M.; Engbersen, J. F. J.; Reinhoudt, D. N., Self-Assembled Monolayers of

Heptapodant β-Cyclodextrins on Gold. Langmuir 1998, 14, 6424-6429.

(33) Liu, W.; Zhang, Y.; Gao, X., Interfacial Supramolecular Self-Assembled Monolayers of C60 by

Thiolated β-Cyclodextrin on Gold Surfaces Via Monoanionic C60. J. Am. Chem. Soc. 2007, 129,

4973-4980.

(34) Rojas, M. T.; Koeniger, R.; Stoddart, J. F.; Kaifer, A. E., Supported Monolayers Containing

Preformed Binding Sites. Synthesis and Interfacial Binding Properties of a Thiolated β-Cyclodextrin

Derivative. J. Am. Chem. Soc. 1995, 117, 336-343.

(35) Méndez-Ardoy, A.; Steentjes, T.; Kudernac, T.; Huskens, J., Self-Assembled Monolayers on

Gold of β-Cyclodextrin Adsorbates with Different Anchoring Groups. Langmuir 2014, 30, 3467-

3476.

(36) Beulen, M. W. J.; Bugler, J.; De Jong, M. R.; Lammerink, B.; Huskens, J.; Schönherr, H.; Vancso,

G. J.; Boukamp, B. A.; Wieder, H.; Offenhäuser, A.; Knoll, W.; Van Veggel, F. C. J. M.; Reinhoudt, D.

N., Host-Guest Interactions at Self-Assembled Monolayers of Cyclodextrins on Gold. Chem. - Eur.

J. 2000, 6, 1176-1183.

(37) Godínez, L. A.; Schwartz, L.; Criss, C. M.; Kaifer, A. E., Thermodynamic Studies on the

Cyclodextrin Complexation of Aromatic and Aliphatic Guests in Water and Water−Urea Mixtures.

Experimental Evidence for the Interaction of Urea with Arene Surfaces. J. Phys. Chem. B 1997, 101,

3376-3380.

(38) Mulder, A.; Auletta, T.; Sartori, A.; Del Ciotto, S.; Casnati, A.; Ungaro, R.; Huskens, J.;

Reinhoudt, D. N., Divalent Binding of a Bis(Adamantyl)-Functionalized Calix[4]Arene to Β-

Cyclodextrin-Based Hosts: An Experimental and Theoretical Study on Multivalent Binding in

Solution and at Self-Assembled Monolayers. J. Am. Chem. Soc. 2004, 126, 6627-6636.

(39) Huskens, J.; Mulder, A.; Auletta, T.; Nijhuis, C. A.; Ludden, M. J. W.; Reinhoudt, D. N., A Model

for Describing the Thermodynamics of Multivalent Host−Guest Interactions at Interfaces. J. Am.

Chem. Soc. 2004, 126, 6784-6797.



(41) Clark, R. A.; Bowden, E. F., Voltammetric Peak Broadening for Cytochrome C/Alkanethiolate

Monolayer Structures: Dispersion of Formal Potentials. Langmuir 1997, 13, 559-565.

(42) Thoden van Velzen, E. U.; Engbersen, J. F. J.; Reinhoudt, D. N., Self-Assembled Monolayers of

Receptor Adsorbates on Gold: Preparation and Characterization. J. Am. Chem. Soc. 1994, 116,

3597-3598.



(44) Nkosi, D.; Pillay, J.; Ozoemena, K. I.; Nouneh, K.; Oyama, M., Heterogeneous Electron Transfer

Kinetics and Electrocatalytic Behaviour of Mixed Self-Assembled Ferrocenes and Swcnt Layers.

Phys. Chem. Chem. Phys. 2010, 12, 604-613.


83

(45) Henke, C.; Steinem, C.; Janshoff, A.; Steffan, G.; Luftmann, H.; Sieber, M.; Galla, H.-J., Self-

Assembled Monolayers of Monofunctionalized Cyclodextrins onto Gold: A Mass Spectrometric

Characterization and Impedance Analysis of Host−Guest Interaction. Anal. Chem. 1996, 68, 3158-

3165.

(46) Laviron, E., General Expression of the Linear Potential Sweep Voltammogram in the Case of

Diffusionless Electrochemical Systems. J. Electroanal. Chem. Interfacial Electrochem. 1979, 101,

19-28.

(47) Roth, K. M.; Yasseri, A. A.; Liu, Z.; Dabke, R. B.; Malinovskii, V.; Schweikart, K.-H.; Yu, L.;

Tiznado, H.; Zaera, F.; Lindsey, J. S.; Kuhr, W. G.; Bocian, D. F., Measurements of Electron-Transfer

Rates of Charge-Storage Molecular Monolayers on Si(100). Toward Hybrid

Molecular/Semiconductor Information Storage Devices. J. Am. Chem. Soc. 2003, 125, 505-517.

(48) Weber, K.; Hockett, L.; Creager, S., Long-Range Electronic Coupling between Ferrocene and

Gold in Alkanethiolate-Based Monolayers on Electrodes. J. Phys. Chem. B 1997, 101, 8286-8291.

(49) Bueno, P. R.; Mizzon, G.; Davis, J. J., Capacitance Spectroscopy: A Versatile Approach to

Resolving the Redox Density of States and Kinetics in Redox-Active Self-Assembled Monolayers. J.

Phys. Chem. B 2012, 116, 8822-8829.

(50) Góes, M. S.; Rahman, H.; Ryall, J.; Davis, J. J.; Bueno, P. R., A Dielectric Model of Self-

Assembled Monolayer Interfaces by Capacitive Spectroscopy. Langmuir 2012, 28, 9689-9699.

(51) Fabre, B.; Ababou-Girard, S.; Singh, P.; Kumar, J.; Verma, S.; Bianco, A., Noncovalent Assembly

of Ferrocene on Modified Gold Surfaces Mediated by Uracil–Adenine Base Pairs. Electrochem.

Commun. 2010, 12, 831-834.

(52) Smalley, J. F.; Newton, M. D.; Feldberg, S. W., A Simple Comparison of Interfacial Electron-

Transfer Rates for Surface-Attached and Bulk Solution-Dissolved Redox Moieties. J. Electroanal.

Chem. 2006, 589, 1-6.

(53) Lagrost, C.; Alcaraz, G.; Bergamini, J.-F.; Fabre, B.; Serbanescu, I., Functionalization of Silicon

Surfaces with Si-C Linked β-Cyclodextrin Monolayers. Chem. Commun. 2007, 1050-1052.

(54) Mulder, A.; Onclin, S.; Péter, M.; Hoogenboom, J. P.; Beijleveld, H.; ter Maat, J.; García-Parajó,

M. F.; Ravoo, B. J.; Huskens, J.; van Hulst, N. F.; Reinhoudt, D. N., Molecular Printboards on Silicon

Oxide: Lithographic Patterning of Cyclodextrin Monolayers with Multivalent, Fluorescent Guest

Molecules. Small 2005, 1, 242-253.

Chapter 4

84

85

Chapter 5

Electron transfer processes in ferrocene-modified

poly(ethylene glycol) monolayers on electrodes

Electrochemistry is a powerful tool to study self-assembled monolayers. Here,

we modified electrodes with different lengths of linear poly(ethylene glycol)

(PEG) polymers end-functionalized with a redox-active ferrocene (Fc) group. The

electron transport properties of the Fc probes were studied using cyclic

voltammetry. The Fc moiety attached to the shortest PEG (Mn=250 Da) behaved

as a surface-confined species, and the homogeneous electron transfer rate

constants were determined. The electron transfer of the ferrocene group on the

longer PEGs (Mn=3.4, 5 and 10 kDa) was shown to be driven by diffusion. For low

surface densities, where the polymer exists in the mushroom conformation, the

diffusion coefficients (D) and rate constants were increasing with polymer

length. In the loose brush conformation, where the polymers are close enough

to interact with each other, the thickness of the layers (e) was unknown and a

parameter D1/2/e was determined. This parameter showed no dependence on

surface density and an increase with polymer length.

Chapter 5

86

5.1 Introduction

Understanding conformations of (bio)polymers at surfaces is important since

such layers are widely used for electrochemical detection methods, for example

for DNA sensing.1-7 The mechanism of so-called E-DNA sensors relies on changes

in conformation upon binding with the analyte, and this conformational change

results in a change in signal.3 As such, the flexibility of the linker plays an

important role in the signal output.

The configuration of end-tethered polymers on surfaces is dictated by the

surface density and the length of the polymers.8-9 The transition between the

mushroom and brush conformation, where the polymers begin to overlap has

been modelled10-11 and observed experimentally12-13 with different results for

the onset of the brush formation. If the molecules are tethered with an

electrochemical probe, the behavior of the end-group can be studied using

electrochemical methods. Several studies have been performed with

poly(ethylene glycol) (PEG) layers end-tethered with ferrocene, both with an

atomic force/scanning electrochemical microscope setup14 and on single

electrodes.15-16 The polymer behavior in the mushroom configuration, where the

polymer chains exist as isolated entities, has been modelled.17 These studies

have shown that the electrochemical response of the end-group involves

diffusion to the surface. As the surface density is increased, which brings about

conformational changes, changes in bounded diffusion and kinetics can be

expected, as have indeed been observed for DNA and PNA layers.18 A detailed

experimental study on a well-defined model system in which the length and

density of surface-attached PEG chains are varied is however currently lacking.

Herein we study the effect of polymer length and surface density of end-

tethered PEG layers immobilized on electrodes, in order to study the electron

transfer behavior of the end-group. The surface densities are varied to achieve

the mushroom and loose brush conformations, and the influence of the varying

surface density on the electrochemical properties of the ferrocene group is

established using cyclic voltammetry.


PEG chains bearing on one end a ferrocene (Fc) moiety, used for electrochemical

detection, and on the other end a reactive succinimide (NHS) group, used for

surface attachment, were synthesized according to known procedures.15, 19 Fc-

Electron transfer processes in ferrocene-modified PEG monolayers

87

PEG-NHS derivatives of four molecular weights (Mn = 0.25, 3.4, 5 and 10 kDa, i.e.

Fc-PEG250-NHS, Fc-PEG3k-NHS, Fc-PEG5k-NHS, and Fc-PEG10k-NHS, respectively)

were separately grafted onto gold electrodes that were pretreated with

cystamine. The PEGs (with the exception of PEG250) used here were reported to

have a polydispersity index of 1.08, and their average molecular weights

correspond to fully extended lengths of L= 2.1 nm, 27 nm, 40 nm and 79 nm

(Table 5.1) for PEG250, PEG3k, PEG5k and PEG10k, respectively. The conformation

of the polymers is dictated by the surface density (Figure 5.1). If the density is

sufficiently low (Table 5.1), the polymers exhibit a mushroom conformation. As

the surface density increases and the chains start to interact, the polymers

extend outwards into the solution and form a loose brush.

Figure 5.1: Surface density effect on polymer conformation. If the surface density is sufficiently low, i.e. when the Flory radius (Rf) is smaller than the distance between the grafting points (s), the polymers are in the mushroom conformation (left). When the surface density is increased, the polymers start to overlap, and a loose brush is formed (right).

Table 5.1: Overview of the used polymers, their molecular weights and standard deviations calculated from the reported polydispersity (1.08), calculated Flory radius, Rf, fully extended chain length (L), and the calculated surface density at which the chains start to interact and move into a loose brush regime (for the calculations of these densities, see the Experimental).

Mn (kDa) Rf (nm) L (nm) Mushroom to brush transition

density (mol/cm2)

0.25* 1.0 2.1 1.6 × 10-10

3.4 ± 1.0 4.7 27 7.4 × 10-12

5.0 ± 1.4 6.0 40 4.6 × 10-12

10 ± 2.8 9.1 79 2.0 × 10-12

*Polydispersity not reported.

Chapter 5

88

Cleaned gold electrodes were modified with cystamine, resulting in a monolayer

with free amine groups onto which the Fc-PEG-NHS polymers were grafted via

their succinimide groups. Cyclic voltammetry measurements of the modified

electrodes were performed in a 1 M NaClO4 solution. The results, see Figure 5.2a

for a cyclic voltammogram of a the Fc-PEG250 layer, were typical for surface-

attached species, with a small peak separation (<59 mV) and a peak current that

was linearly dependent on the scan rate, indicating that all the redox couples

have sufficient time to contribute to the electron transfer. In this regime, the

charge determined from the peak area remains constant independent of scan

rate, and the surface density (Γ) of the PEG molecules can be determined

according to Equation 5.1.

Γ = Q/nFA (Equation 5.1)

In Equation 5.1, Q is the charge of the integrated peak area, A the microscopic

surface area as determined from sulfuric acid cleaning scans, F the Faraday

constant, and n the number of electrons involved (n=1 for ferrocene). At scan

rates above 200 V/s a significant peak separation became apparent for Fc-

PEG250, resulting in a characteristic trumpet plot (Figure 5.2b) when plotting the

peak separation, η, versus the logarithm of the scan rate, ν.

Figure 5.2: a) Cyclic voltammogram of a Fc-PEG250 monolayer, obtained upon a reaction time of 5

min, recorded in 1 M NaClO4 at 2 V/s vs a Hg/Hg2SO4 reference electrode. b) Trumpet plot of the

peak separation, η, versus the logarithm of the scan rate, ν.

When the peak separation increased past 200 mV and the electron transfer

became electrochemically irreversible, the peak potentials changed linearly with

the logarithm of the scan rate. In that case, a standard rate constant, k, can be

determined using Laviron’s formulation, Equation 5.2.20


89

k=𝛼𝑛𝐹𝜈c

𝑅𝑇=

(1−𝛼)𝑛𝐹𝜈a

𝑅𝑇 (Equation 5.2)

In Equation 5.2, α is the transfer coefficient, taken as 0.5, and R and T are the

ideal gas constant and the temperature, respectively, while νc and νa are the

cathodic and anodic scan rates, respectively. From this treatment, rate

constants k of 2.4 × 103 s-1 and 1.7 × 103 s-1 were found for the anodic and

cathodic processes, respectively. These values are significantly larger than the

standard rate constants determined for ferrocene moieties on well packed

alkane monolayers of similar length,21 thus indicating a loosely packed layer in

the case of PEG layers. This is confirmed by the measured surface densities, for

which values between 2 and 3 × 10-10 mol/cm2 were obtained when using a

surface functionalization time of 5 min.

The peak separation for the longer PEGs was less pronounced and was mostly

caused by a shift of the cathodic peak and a smaller shift of the anodic peak

combined with significant peak broadening (Figure 5.3a and d). At increased

scan rates, the current became linear with the square root of the scan rate

(Figure 5.3c) following the Randles-Sevcik equation,22 indicating an additional

dependence of the electron transfer rate on the diffusion of the ferrocene head

groups to and from the surface. Since the effect of diffusion on the electron

transfer excludes the use of the Laviron method for the determination of rate

constants, these were determined using the Nicholson method for diffusing

species adjusted for surface-confined species.18, 23-24 In order to do this, the

diffusion constants of ferrocene and oxidized ferrocenium species (DFc and DFc+,

respectively) were determined from the anodic and cathodic peak currents

using the Randles-Sevcik equation (Equation 5.3).

𝑖p = 0.4463𝑛𝐹𝐴𝐶 (𝑛𝐹𝜈𝐷

𝑅𝑇)

1

2 (Equation 5.3)

In Equation 5.3, the concentration, C, can be replaced by Γ/e, with e being the

layer thickness. Normalizing the anodic peak current densities (ja = ia/A) to ν1/2

showed that at higher scan rates ja/ν1/2 became independent of the scan rate,

thus reaching a plateau (Figure 5.3c). From the height of this plateau the

diffusion coefficients DFc and DFc+ were determined using the Randles-Sevcik

equation for the polymers in the mushroom conformation. When the surface

density is sufficiently low so that the polymers on the surface can be assumed

to exist in the mushroom conformation (see Table 5.1) and do not interact with

Chapter 5

90

each other, the average thickness of the polymer layer, e, can be taken as the

Flory radius.

Figure 5.3: a) Cyclic voltammogram of a 1.5 x 10-11 mol/cm2 monolayer of Fc-PEG10k (obtained

from a reaction time of 90 min) in 1 M NaClO4 at 10 V/s. b) Anodic peak current, ia, plotted versus

the square root of the scan rate ν; the line is a linear fit to the data. c) Anodic peak current density

(ja = ia/A), normalized to the square root of the scan rate, vs the logarithm of the scan rate. d) Peak

separation vs the logarithm of the scan rate. e) The obtained surface densities as a function of the

reaction time, t.

As can be seen in Table 5.2, the determined diffusion coefficients show that the

bound ferrocenium cation diffuses faster (2.5 - 4 times) than the reduced

ferrocene, which is also visible from the peak asymmetry (Figure 5.3a), the

cathodic peak being sharper than the anodic peak.15 This effect is not present

for ferrocene species in solution25-26 but has been noted before for surface-

bound Fc-PEG, and has been attributed to the positively charged Fc+ being


91

‘propelled away’ from the surface, caused by electrostatic repulsion between

the surface and the Fc+ moiety directly after the oxidation step.15

The diffusion coefficients increased with increasing polymer length, which is

counterintuitive. For the mushroom configuration, the concentration of

ferrocene can be represented by a Gaussian distribution with the highest

concentration close to the surface. The longer molecules have a higher local

density due to the amount of polymer surrounding the ferrocene head, which is

expected to slow down diffusion. Indeed, the opposite trend has been observed

for different lengths of Fc-modified PNA strands tethered on electrodes, with

the diffusion coefficients decreasing and the system becoming more surface

confined as the strand length decreased.18 On the other hand, it has been shown

that the diffusion coefficient for tethered PEG3k is higher than for PEG600 in

dichloromethane, even with the PEG3k being in a loose brush conformation and

the PEG6k in a mushroom conformation, which has been attributed to an

increased influence of the spring constant on the diffusion coefficient.15

Table 5.2: Diffusion coefficients and homogeneous electron transfer rate constants determined

for Fc-PEG3k, Fc-PEG5k and Fc-PEG10k in the mushroom regime.

polymer DFc (× 10-12

cm2/s) DFc+ (× 10-12

cm2/s)

k0 (s-1)

Fc-PEG3k 1.4 ± 1.0 6.0 ± 1.3 88 ± 23

Fc-PEG5k 1.9 ± 0.4 6.2 ± 0.4 114 ± 104

Fc-PEG10k 9.1 ± 6.3 29.8 ± 9.7 228 ± 54

As mentioned above, using the diffusion coefficients, the homogeneous

electron transfer rate constants could be determined using the Nicholson

method for diffusing species when the peak separation is between 61 and 212

mV. In short, the peak separation is related by a kinetic parameter ψ, from which

the rate constant can be calculated.23 As can be seen in Table 5.2, the

determined rate constants are lower than the value (approx. 2 × 103 s-1)

determined for PEG250, in which case the electron transfer is not affected by

diffusion. Furthermore, the rate constants increased with increasing polymer

length, which could indicate that for the longer molecules the ferrocene end

groups are on average closer to the surface.18

Chapter 5

92

As the surface density was increased by employing longer reaction times of Fc-

PEG-NHS on the cystamine surfaces (Figure 5.4a), the average distance between

the anchoring points became smaller than the Flory radius, and consequently

the polymer conformation moved from a mushroom into a loose brush regime.

In the brush regime, the polymer chains stretch outward into the solution,

therefore, the layer thickness (e) is no longer related to the Flory radius, but

instead becomes proportional to the average number, N, of monomers per

chain, by e ~ pNaσ1/3,8 where a is the monomer size, σ the graft density and p a

proportionality coefficient. Since the actual layer thickness is unknown in this

case, the diffusion and rate constants cannot be directly determined.

Figure 5.4: a) The obtained surface densities as a function of the reaction time, t. b) and c) The

anodic (ja) and cathodic (jc) peak current densities normalized to the square root of the scan rate,

vs surface density, for Fc-PEG3k, Fc-PEG5k and Fc-PEG10K. d) The Gaussian fully extended chain

length distribution for PEG3k, PEG5k and PEG10k (represented in all graphs by the colors red, blue

and black, respectively).

The anodic and cathodic peak current densities normalized to the square root

of ν, jc/ν1/2, showed a linear increase upon increasing surface density (Figure 5.4b


93

and c). Since the slope of this line is now solely dependent on the parameter

DFc+1/2/e (see Equation 5.3), this parameter was determined for all three PEG

lengths in the brush conformation. The values for DFc+1/2/e were found to be 10.6

± 1.7, 9.2 ± 0.6 and 9.1 ± 0.8 s-1/2 for Fc-PEG3k, Fc-PEG5k, and Fc-PEG10k,

respectively, when using the cathodic peak current densities. The DFc1/2/e values,

determined from the anodic peak current densities, were again lower, 4.5 ± 1.4,

6.6 ± 0.7, and 6.1 ± 1.5 s-1/2 . The similarity between the PEGs can partly be

attributed to the broad size distributions, which show that a large proportion of

the size distributions overlap (Figure 5.4d), despite the difference in Mn and a

relatively small polydispersity of 1.08. The fact that the D1/2/e values remain

constant implies that, as the layer thickness increases, the diffusion constants

must increase as well. An increase of the diffusion constant with increased

surface density can be explained with an increased proximity of the redox

centers to the surface.27 For a surface density of 1.5 × 10-12 mol/cm2, and a layer

thickness of 40 nm (half of the fully extended length of PEG10k), the ferrocene

concentration inside that layer can be calculated to be 3.8 mM, which is of the

same order of magnitude as electrochemical mediators in solution.28 This cannot

be the sole contribution to the diffusion process, since the diffusion constant is

greater for PEG10k, which must mean that the local ferrocene concentration is

lower than for shorter PEGs, as the layer thickness is larger.

5.3 Conclusions

Surfaces were modified with different lengths of poly(ethylene glycol) (PEG)

end-tethered with a ferrocene moiety and studied using cyclic voltammetry. The

shortest Fc-PEG, Fc-PEG250, behaved as a surface-confined layer for which

homogeneous rate constants could be determined using the Laviron method.

These results suggest that a loosely packed layer was formed. For longer Fc-

PEGs, diffusion coefficients and homogeneous electron transfer rate constants

could be determined when the layers were present in the mushroom

conformation, both of which increased with the polymer length. As the surface

density was increased, and the polymer layers entered the loose brush regime,

the determination of these parameters was not possible due to the unknown

layer thickness. Instead, D1/2/e values were determined for both ferrocene and

ferrocenium, and were shown to be constant for increasing surface densities,

implying an increase in diffusion coefficient with surface density. This increase

is attributed to an increased chance of electron hopping as the local ferrocene

Chapter 5

94

concentration increases. However, the diffusion coefficient also appears to

become larger for longer PEGs, while these form a thicker layer and thus a lower

Fc concentration. More information is needed to fully explain this increase.

Overall, the data presented here highlight that redox-modified polymers

attached to an electrode surface show a rich and complex electrochemical

behavior, that leads to currents that depend on a multitude of parameters.

Unraveling these dependencies will assist in the development of

electrochemical sensors that rely on redox behavior of surface-tethered probe

molecules.


5.4.1 Materials

Reagents and solvents were purchased from Sigma-Aldrich, high-purity water

(MilliQ) was used (Millipore, R = 18.2 Ω). All bis-NHS-functionalized PEGs were

purchased from Nanocs and have a reported dispersity of 1.08. The Fc-PEG-NHS

molecules were synthesized according to known procedures.15 Fc-PEG250-NHS

was further purified by column chromatography with dichloromethane as

eluent. After the first fraction was removed, the eluent was changed to

DCM/EtOH (95:5). Fc-PEG3k-NHS, Fc-PEG5k-NHS, and Fc-PEG10k-NHS was further

purified by size exclusion chromatography (Biobeads SX-1) with DCM as eluent.

2-Ferrocene-ethylamine

2-Ferrocene-ethylamine was synthesized by the reduction of ferrocene

acetonitrile by LiAlH4 as described in the literature,19 with some modifications.

An amount of 0.25 g (6.5 mmol) LiAlH4 and 0.6 g (4.5 mmol) AlCl3 were carefully

added to 10 mL dry THF while stirring in an ice bath. Ferrocene acetonitrile (0.5

g, 2.25 mmol) was dissolved in 5 mL dry THF and subsequently added to the

cooled mixture and refluxed overnight under an argon atmosphere. After

cooling, water was added drop wise to decompose the excess LiAlH4. A volume

of 0.25 mL of concentrated NaOH was added to destroy the formed AlCl3/2-

ferrocene-ethylamine complex. The aqueous phase was extracted three times

with diethylether. The combined organic phases were dried with MgSO4 and

filtered, and the solvent was removed by rotary evaporation. The product was

purified by column chromatography with dichloromethane as the eluent. After

drying in vacuo a brown solid was obtained (0.13 g; 26%). 1H NMR (300 MHz,

CDCl3): δ(ppm) 4.2 (m, 9H, Fc), 2.82 (t, 2H, CH2-Fc), 2.48 (t, 2H, CH2-N).


95

PEG250-(NHS)2

The bis-NHS ester of the PEG250 diacid was prepared according to a procedure

described earlier.15 An amount of 0.44 g (3.8 mmol) of N-hydroxysuccinimide

(NHS) and 0.78 g (3.8 mmol) dicyclohexylcarbodiimide (DCC) were added to a

stirred solution of 0.4 g (1.6 mmol) poly(ethylene glycol) bis(carboxymethyl)

ether in 30 mL 1,4-dioxane. After stirring over night at room temperature under

an argon atmosphere, the mixture was filtered to remove the 1,3-

dicyclohexylurea precipitate. The solvent was removed by rotary evaporation

and the residue was further dried under vacuum. 1H NMR (300 MHz, CDCl3):

δ(ppm) = 4.56 (s, 4H, CH2C=O), 366 (s, 12H, C2H4-O), 2.81 (s, 8H, NHS).

5.4.2 Surface functionalization and electrochemistry

All electrochemical measurements were performed on a CH Instruments

bipotentiostat 760D. Cyclic voltammetry measurements were performed in a

three-electrode setup using 2 mm diameter (CH Instruments) or 1.6 mm

diameter (BASi) gold disk electrodes, a platinum wire as the counter electrode

and a Ag/AgCl reference electrode in aqueous 1 M NaClO4.

Before modification the electrodes were polished using 50 nm alumina particles

(CH Instruments), followed by extensive rinsing with ethanol and 5 min of

ultrasonic treatment in ethanol and 5 min in MilliQ water. Subsequently, the

electrodes were cleaned electrochemically in 0.5 M H2SO4 by applying an

oxidizing potential of 2 V for 5 seconds followed by a reducing potential of -0.35

V for 10 s. Then the electrode potential was scanned from -0.25 V to 1.55 V and

back for 40 cycles at a scan rate of 100 mV/s, the surface area of the electrodes

was determined from the Au-O reduction peak, using a value of 386 µC/cm2 to

convert the charge into surface area.29-30

After cleaning, the electrodes were rinsed with MilliQ water and ethanol and

dried under a flow of N2 and placed immediately in a 2 mM cystamine solution

and left over night. Subsequently, the electrodes were rinsed with MilliQ water

and dried under a flow of N2 and placed in a 0.2 mM Fc-PEG-NHS solution. The

surface densities were varied by changing the reaction time between 5-120 min.

The prepared electrodes were copiously rinsed with MilliQ water and

subsequently cycled electrochemically at 100 mV/s in 1 M NaClO4 until a stable

signal was obtained.

Chapter 5

96

5.4.3 Calculations

The Flory radii (Rf) in Table 5.1 were calculated with Rf = aN3/5 where a is the size

of the individual ethylene glycol monomer (a = 0.35 nm31) and N the degree of

polymerization (N = 6, 77, 114 and 227 for PEG250, PEG3k PEG5k and PEG10k,

respectively). When the distance between the grafting points (s) of the PEG

molecules approaches the Rf, the chains start to overlap and form a polymer

brush at the critical dimensionless coverage σcritical = (a/Rf)2 = (NaΓ)a2, where Na

is Avogadro’s constant. From this the critical surface densities as shown in Table

5.1 can be determined.14

5.5 References



A. 2003, 100, 9134-9137.

(2) Prieto-Simón, B.; Campàs, M.; Marty, J. L., Electrochemical Aptamer-Based Sensors.

Bioanalytical Reviews 2010, 1, 141-157.

(3) Ricci, F.; Plaxco, K. W., E-DNA Sensors for Convenient, Label-Free Electrochemical Detection of

Hybridization. Microchim. Acta 2008, 163, 149-155.

(4) Xiang, Y.; Qian, X.; Jiang, B.; Chai, Y.; Yuan, R., An Aptamer-Based Signal-on and Multiplexed

Sensing Platform for One-Spot Simultaneous Electronic Detection of Proteins and Small

Molecules. Chem. Commun. 2011, 47, 4733-4735.

(5) Xiao, Y.; Qu, X.; Plaxco, K. W.; Heeger, A. J., Label-Free Electrochemical Detection of DNA in

Blood Serum Via Target-Induced Resolution of an Electrode-Bound DNA Pseudoknot. J. Am. Chem.

Soc. 2007, 129, 11896-11897.

(6) Xu, X.; Li, B.; Xie, X.; Li, X.; Shen, L.; Shao, Y., An I-DNA Based Electrochemical Sensor for Proton

Detection. Talanta 2010, 82, 1122-1125.

(7) Zhang, K.; Zhu, X.; Wang, J.; Xu, L.; Li, G., Strategy to Fabricate an Electrochemical Aptasensor:

Application to the Assay of Adenosine Deaminase Activity. Anal. Chem. 2010, 82, 3207-3211.

(8) De Gennes, P. G., Conformations of Polymers Attached to an Interface. Macromolecules 1980,

13, 1069-1075.

(9) Alexander, S., Adsorption of Chain Molecules with a Polar Head. A Scaling Description. J. Phys.

France 1977, 38, 983-987.

(10) Pépin, M. P.; Whitmore, M. D., Monte Carlo and Numerical Self-Consistent Field Study of End-

Tethered Polymers in Good Solvent. J. Chem. Phys. 1999, 111, 10381-10388.

(11) Karaiskos, E.; Bitsanis, I. A.; Anastasiadis, S. H., Monte Carlo Studies of Tethered Chains. J.

Polym. Sci., Part B: Polym. Phys. 2009, 47, 2449-2461.


97

(12) Wu, T.; Efimenko, K.; Genzer, J., Combinatorial Study of the Mushroom-to-Brush Crossover in

Surface Anchored Polyacrylamide. J. Am. Chem. Soc. 2002, 124, 9394-9395.

(13) Dumont, E. L. P.; Belmas, H.; Hess, H., Observing the Mushroom-to-Brush Transition for

Kinesin Proteins. Langmuir 2013, 29, 15142-15145.

(14) Abbou, J.; Anne, A.; Demaille, C., Probing the Structure and Dynamics of End-Grafted Flexible

Polymer Chain Layers by Combined Atomic Force-Electrochemical Microscopy. Cyclic

Voltammetry within Nanometer-Thick Macromolecular Poly(Ethylene Glycol) Layers. J. Am. Chem.

Soc. 2004, 126, 10095-10108.







(17) Abbou, J.; Anne, A.; Demaille, C., Accessing the Dynamics of End-Grafted Flexible Polymer

Chains by Atomic Force-Electrochemical Microscopy. Theoretical Modeling of the Approach

Curves by the Elastic Bounded Diffusion Model and Monte Carlo Simulations. Evidence for

Compression-Induced Lateral Chain Escape. J. Phys. Chem. B 2006, 110, 22664-22675.



Eur. J. 2011, 17, 9678-9690.

(19) Seiwert, B.; Karst, U., Analysis of Cysteine-Containing Proteins Using Precolumn Derivatization

with N-(2-Ferroceneethyl)Maleimide and Liquid Chromatography/Electrochemistry/ Mass

Spectrometry. Anal. Bioanal. Chem. 2007, 388, 1633-1642.

(20) Laviron, E. E. E., General Expression of the Linear Potential Sweep Voltammogram in the Case

of Diffusionless Electrochemical Systems. J. Electroanal. Chem. Interfacial Electrochem. 1979, 101,

19-28.







(24) Anne, A.; Demaille, C., Dynamics of Electron Transport by Elastic Bending of Short DNA

Duplexes. Experimental Study and Quantitative Modeling of the Cyclic Voltammetric Behavior of

3′-Ferrocenyl DNA End-Grafted on Gold. J. Am. Chem. Soc. 2006, 128, 542-557.

Chapter 5

98

(25) Martin, R. D.; Unwin, P. R., Theory and Experiment for the Substrate Generation/Tip

Collection Mode of the Scanning Electrochemical Microscope: Application as an Approach for

Measuring the Diffusion Coefficient Ratio of a Redox Couple. Anal. Chem. 1998, 70, 276-284.

(26) Mampallil, D.; Mathwig, K.; Kang, S.; Lemay, S. G., Redox Couples with Unequal Diffusion

Coefficients: Effect on Redox Cycling. Anal. Chem. 2013, 85, 6053-6058.

(27) Akhoury, A.; Bromberg, L.; Hatton, T. A., Interplay of Electron Hopping and Bounded Diffusion

During Charge Transport in Redox Polymer Electrodes. J. Phys. Chem. B 2013, 117, 333-342.



1485-1492.

(29) Trasatti, S.; Petrii, O. A., Real Surface Area Measurements in Electrochemistry. J. Electroanal.

Chem. 1992, 327, 353-376.

(30) Tremiliosi-Filho, G.; Dall'Antonia, L. H.; Jerkiewicz, G., Limit to Extent of Formation of the

Quasi-Two-Dimensional Oxide State on Au Electrodes. J. Electroanal. Chem. 1997, 422, 149-159.

(31) Allen, C.; Dos Santos, N.; Gallagher, R.; Chiu, G. N. C.; Shu, Y.; Li, W. M.; Johnstone, S. A.;

Janoff, A. S.; Mayer, L. D.; Webb, M. S.; Bally, M. B., Controlling the Physical Behavior and Biological

Performance of Liposome Formulations through Use of Surface Grafted Poly(Ethylene Glycol).

Biosci. Rep. 2002, 22, 225-250.

99

Chapter 6

Electron transfer mediated by surface-tethered redox

groups in nanofluidic devices

Electrochemistry provides a powerful sensor transduction and amplification

mechanism that is highly suited for use in integrated, massively parallelized

assays. Here we demonstrate the cyclic voltammetric detection of flexible, linear

poly(ethylene glycol) polymers that have been functionalized with redox-active

ferrocene (Fc) moieties and surface-tethered inside a nanofluidic device

consisting of two microscale electrodes separated by a gap of <100 nm. Diffusion

of the surface-bound polymer chains in the aqueous electrolyte allows the redox

groups to repeatedly shuttle electrons from one electrode to the other, resulting

in a greatly amplified steady-state electrical current. Variation of the polymer

length provides control over the current, as the activity per Fc moiety appears to

depend on the extent to which the polymer layers of the opposing electrodes can

interpenetrate each other and thus exchange electrons. These results outline the

design rules for sensing devices that are based on changing the polymer length,

flexibility and/or diffusivity by binding an analyte to the polymer chain. Such a

nanofluidic enabled configuration provides an amplified and highly sensitive

alternative to other electrochemical detection mechanisms.

This chapter has been published in: T. Steentjes, S. Sarkar, P. Jonkheijm, S. G. Lemay, J. Huskens,

Small 2017, 13, 1603268.

Chapter 6

100

6.1 Introduction

Biosensing plays an increasingly important role, in particular in the development

of personalized medicine.1 This growing importance also puts new demands on

the performance of such devices, which ranges from higher sensitivities and

selectivities, to the use of smaller sample volumes, the use of body fluids, an

easy readout and signal processing, and the option to use disposable devices.

Electrochemical devices have particular advantages that meet several of these

requirements.2 The combination with electrochemistry opens pathways for a

multitude of benefits for biosensing strategies that allow their use in

personalized medicine.3-5 A key opportunity offered by electrochemistry is an

enhanced sensitivity based on signal amplification, which can be achieved when

multiple electrons, e.g. in cyclic redox processes, amplify a single molecular

recognition event.

Prime examples of sensitive electrochemical detection are redox-activated DNA

(E-DNA) biosensors, which rely on conformational changes that modulate the

motion and the distance of the redox moiety to an electrode, resulting in a

change in signal.6-8 By default, the nature of the linker and the surface density

of the redox-labeled polymer chains play a pivotal role in the optimization of the

sensitivity of such a sensor. Long linear molecular chains such as synthetic

polymers9-11 and DNA strands end-capped with an electrochemically active

label12-14 have been shown to give faradaic currents dictated by the diffusional

motion of the redox moiety. This is in contrast with surface-immobilized systems

in which the redox center is located at a fixed distance from the surface and the

electron transfer rate depends exponentially on the distance to the surface.15-17

A further increase in sensitivity can be achieved by the use of electrochemical

mediators which replenish the oxidized surface layer.18 Re-activation of the

diffusing species in the presence of a second electrode providing an opposing

reducing or oxidizing potential, as is performed in scanning electrochemical

microscopy (SECM) and in nanofluidic devices,19 provides a significant

amplification of the faradaic current due to redox cycling. This concept has been

exploited for biodetection with increased sensitivity.20-21 Alternatively, if the

second electrode is sufficiently close to the surface to get in contact with the

electrochemically active layer, the electrode can take the role of the mediator

by replenishing the oxidized/reduced species directly. Such a strategy has been

employed using a conductive atomic force microscopy (AFM) tip positioned on

Electron transfer mediated by tethered redox groups in nanofluidic devices

101

top of poly(ethylene glycol) (PEG)22 and DNA23 layers, which allows the direct

measurement of the redox cycling current provided by the bounded diffusion of

the electrode-attached redox labels.

Nanodevices with nanometric electrode spacings, comparable to the molecular

lengths of the surface-attached polymer chains, provide an excellent basis for

electrochemical devices in which electron shuttling can occur in the absence of

a mediator. This approach further promises the well-controlled and

reproducible architecture of a closed solid-state device as well as integration

with microfluidics, sample handling, and signal processing.24 Several device

architectures have been demonstrated that lend themselves to miniaturization

down to the nanometer scale, including interdigitated electrode arrays,25-27

recessed ring electrodes28 and thin-layer cells.19, 29-30 As of yet, however, no such

system has been reported in a device configuration where surface-attached

redox couples are directly replenished due to the effect of the second electrode

without the need of a mediator.

In this chapter we report nanofluidic devices with two opposing microscale

electrodes spaced by a sub-100 nm gap and their functionalization with

electrochemically active end-capped polymers in order to study their ability to

transfer electrons between the electrodes across the nanogap in the absence of

a mediator. We compare the effects of different polymer length and length

distribution, as well as the effect of changes in surface density, on the measured

current.


The nanogap devices used in this chapter (Figure 6.1a) consist of two planar

rectangular platinum electrodes that are separated by a distance of 65 nm. To

fabricate the nanofluidic channel, a sacrificial chromium layer was sandwiched

between two platinum electrodes followed by wet etching of the chromium to

open up the nanochannel. Two access holes acted as the inlets to the

nanochannel. The active region for redox cycling is defined by the overlapping

area of the two electrodes, as indicated in Figure 6.1b. Two different types of

nanofluidic devices were used (Type I and Type II), the devices differing solely in

the area of the active region (30 μm2 for Type I and 300 μm2 for Type II). A larger

active region is expected to trap a likewise larger number of redox molecules

between the electrodes, leading to a linear scaling of the current with the area

of the active region. This was confirmed in practice, as described further below.

Chapter 6

102

The platinum electrodes were modified with cystamine, resulting in a monolayer

with exposed amine groups. Onto this monolayer, bifunctional PEG chains were

grafted via a reactive succinimide group present on one end of the chain. The

other end bore a redox-active ferrocene label which was used for

electrochemical detection. Three lengths of PEG, “long” (PEG10k; Mn = 10 ± 2.8

kDa; single standard deviation, polydispersity index PDI = 1.08; average, fully

extended chain length L of 79 nm), “intermediate” (PEG5k; Mn = 5.0 ± 1.4 kDa,

equal PDI; L = 40 nm), and “short” (PEG3k; Mn = 3.4 ± 0.96 kDa, equal PDI; L = 27

nm), were used in this study to probe the possible effects of chain length and

surface density on the current. The PDI causes the polymers to have a rather

broad length distribution (see Chapter 5, Figure 5.4d). Electron transfer is

expected to occur from the reducing electrode onto an oxidized Fc

(ferrocenium) group that, upon diffusion, can transfer the electron to another

Fc group of another PEG chain. Because the polymer lengths used here allow

interpenetration of the chains immobilized at the opposing electrodes, the Fc-

Fc electron transfer may occur from a moiety attached to the reducing electrode

onto one that is immobilized at the oxidizing electrode. Finally, diffusion of a

reduced Fc to the oxidizing electrode allows the transfer of the electron

between them. These electron transfer steps are schematically shown in Figure

6.1c. Differences in polymer length and their coverage on the electrodes are

expected to influence the surface density of polymer and Fc moieties, the

degree of interpenetration of the opposing immobilized layers in the nanogap

device, and possibly the diffusion rate of the Fc moieties within the polymer

layer. These aspects all contribute to the frequencies of electrode-Fc and Fc-Fc

electron transfer events that together define the current.

The nanogap electrodes were initially functionalized with PEG for at least 3 h in

order to obtain maximum surface densities. To make sure that all physisorbed

PEG molecules were removed from the electrodes, the device was held in a

beaker with MilliQ water and vigorously stirred for at least 10 min. Without this

procedure, the redox signal decreased during flow, while the rinsing procedure

resulted in signals that were stable for the duration of the experiments.

Additionally, control experiments, using mercaptoethanol instead of cystamine

thus prohibiting the attachment of the Fc-modified PEG, showed complete

removal of redox activity upon applying the same rinsing method (see

Experimental section, Figure 6.6), and thus confirmed the suitability of this

method.


103

Figure 6.1: a) Schematic diagram of the cross-section of the nanodevice prior to sacrificial layer

etching (not drawn to scale), where z = 65 nm represents the height of the nanogap. b) Optical

microscopy image of the top view of a Type II device before the sacrificial layer was etched. The

dotted white rectangle represents the active area. c) The nanogap electrodes were functionalized

with ferrocene end-capped PEG chains. The PEG layers at opposing electrodes are expected to

interpenetrate to allow a current to flow from the reducing to the oxidizing electrode by a

sequence of electrode-Fc, Fc-Fc, and Fc-electrode electron transfer steps. Reduced ferrocene

groups are depicted in purple, oxidized ferrocenium cation moieties in red, and electron transfer

steps in green.

In order to measure the surface densities at the electrodes, the top and bottom

electrodes were connected and cycled as one electrode as shown in Figure

6.2a.31 The results are illustrated in Figure 6.2b, which shows a cyclic

voltammogram measured on a Type I device grafted with PEG10k. The recorded

voltammogram is characteristic for surface-attached species, with a peak

separation of less than 59 mV for all scan rates employed, a formal potential

(E0ˊ) of 0.3 V vs. Ag/AgCl, and a peak current (Ip) that increases linearly with the

scan rate (Figure 6.2c).32 For surface-attached species, the charge under the

peak is proportional to the total number of electrons transferred and can be

used to calculate the surface density Γ using Γ = Q/nF(Atop + Abottom). Here Q is

the measured charge (as shown in Figure 6.2b), n the number of electrons

transferred per ferrocene moiety (n = 1), F the Faraday constant, and Atop and

Abottom are the geometric surface areas of the two electrodes exposed to the

Chapter 6

104

solution. A surface density of 19 pmol cm-2 was calculated in this case,

corresponding to an average spacing of 3 nm between the polymer chains. The

latter value is smaller than the calculated Flory radius (Rf = 9.1 nm) of the

polymer coils; the value of Rf for good solvents can be calculated from Rf = aN3/5,

where a = 0.35 nm is the monomer length33 and N = 227 is the degree of

polymerization. Therefore it can be concluded that the polymer was attached to

the surface in a loose brush conformation, extending upwards from the

electrode surface.22, 34

Figure 6.2: a) Schematic diagram of the measurement configuration when both electrodes are

cycled together as one electrode. b) Cyclic voltammogram of ferrocene end-capped PEG10k

functionalized electrodes measured in a Type I device at both electrodes simultaneously in a 1 M

NaClO4 solution (scan rate 20 mV s-1). The inset shows the baseline-corrected anodic peak, the

area of which was integrated to extract the surface charge Q and the corresponding surface

density (Γ = 19 pmol cm-2 in this case). c) Plot of the peak current vs. scan rate, indicating a linear

increase of the peak current with the scan rate.

Subsequently, the two electrodes were addressed separately: a reducing

potential (0 V) was applied to the bottom electrode, while the potential of the

top electrode was cycled between 0 and 0.6 V (Figure 6.3a). When the potential

of the sweeping electrode passed E0ˊ, oxidized ferrocenium species attached to

the PEG molecules could regain an electron, either by diffusion to the reducing

electrode or by electron transfer from a Fc group attached to this electrode; the

opposite being true for reduced species contacting the oxidizing electrode.

Combined with Fc-Fc electron transfer between moieties attached to opposite

electrodes caused by layer interpenetration (Figure 6.1c), these electron

transfer events gave rise to a redox cycling current. The redox cycling currents

measured at the two electrodes are expected to have the same magnitude but

opposite signs. The resulting cyclic voltammograms for PEG10k, measured on a


105

Type II device with a surface density of 8.8 pmol cm-2, are shown in Figure 6.3b,

where the simultaneously measured currents through each of the two working

electrodes are plotted. The current at the electrode being cycled includes a large

capacitive contribution which is marked by a pronounced hysteresis (Figure

6.3b, red curve). This capacitance results in part from the electrical double layer

in regions where the electrode is exposed to the solution as well as from the

capacitance of the connecting wires, which have a relatively large area and are

only protected from the solution by a thin passivating layer of SiN/SiO2/SiN. For

this reason we concentrate our analysis below on the current measured at the

electrode held at a fixed potential, which nicely isolates the redox cycling

component.

Figure 6.3: a) Schematic diagram of the measurement configuration showing that the top

electrode is cycled (red) while the bottom electrode is fixed at 0 V (black). b) Simultaneously

recorded voltammograms for PEG10k (Type II device; Γ = 8.8 pmol cm-2). c) Comparison of the redox

cycling current obtained from PEG10k (data from panel b), PEG5k (Type I device; Γ = 25 pmol cm-2)

and PEG3k (Type II device; Γ = 70 pmol cm-2). All measurements were performed at a scan rate of

50 mV s-1 and the currents were normalized by the area Aactive to facilitate comparison.

Additionally, in order to probe the effect of PEG length, a Type I device was

functionalized with PEG5k and a Type II device with a tenfold larger active area

was used for PEG3k. Figure 6.3c shows the obtained redox cycling currents

normalized to the area of the active region, Aactive, for the three different devices.

The steady state redox cycling current obtained for PEG10k is nearly an order of

magnitude higher than that obtained for PEG5k. This is attributed to the PEG5k

layers at the opposing electrodes having less layer interpenetration and thus a

less frequent Fc-Fc electron transfer between Fc-PEGs immobilized at opposite

electrodes. Only the ferrocene on the sweeping electrode is expected to oxidize,

but since the ferrocene moieties on both electrodes can extend half-way

Chapter 6

106

through the nanogap, the ferrocenes attached to the opposing reducing

electrode can donate electrons and act as a mediator. Since the amount of

ferrocene moieties attached to the electrodes is known (Γ = 25 pmol cm-2 in this

case), a concentration of 7.4 mM ferrocene can be calculated to be present

inside the active region. This is a concentration comparable to mediators used

in SECM for measurements on monolayers.35 PEG3k is even shorter and was not

expected to be able to exchange electrons as the average, fully extended length,

L = 27 nm, does not allow the ferrocene labels attached to the electrode to reach

halfway across the channel. However, at the present PDI of 1.08, corresponding

to a standard deviation of 0.96 kDa (or a total of 4.4 kDa) the length of a

significant fraction of the polymer chains can extend beyond 34 nm, which

exceeds half the gap separation. This makes it plausible that also in this case a

fraction of polymer chains is capable of participating in electron transfer.

Combined with the higher coverage for PEG3k (70 pmol cm-2), this explains the

observed similar current values obtained for PEG5k and PEG3k.

The experiments of Figures 6.2 and 6.3 were repeated in several additional

devices for all three chain lengths, and the results are summarized in Figure 6.4.

The measured surface densities decreased with increasing polymer length

(Figure 6.4a), as expected since the increased Flory radius leads to steric

hindrance. Some scatter is observed in the data, which we attribute primarily to

surface roughness: the area values are based on the geometric surface area and

averaged over both electrodes, whereas the surface roughness is not the same

for both electrodes (see Experimental section) and can vary between devices.

The corresponding redox cycling currents were recorded simultaneously and

were similar to the examples shown in Figure 6.3. To get a clear picture of the

effect of the linker length, the redox cycling currents were normalized to the

surface density according to v = IRC/(eNactive) (with e the charge of an electron

and Nactive the amount of PEG-Fc molecules in the active area) – yielding the rate

of electron transfer per available ferrocene group – as presented in Figure 6.4b

(real currents normalized for the active area are shown in Figure 6.4c). This

analysis shows that a single PEG10k chain functionalized with a ferrocene end-

group is able to transfer on average 6 electrons to the opposing electrode per

second, whereas the electron transfer rate drops to 1 s-1 per ferrocene for PEG5k,

reflecting the decrease in polymer length and the lower frequency of Fc-Fc

electron transfer events between oppositely immobilized chains. It also

becomes clear that, although the measured redox cycling currents for PEG3k are


107

not significantly lower than for PEG5k, the rate of electrons transferred per

ferrocene available is only 0.3 s-1 for PEG3k, and thus the measured redox cycling

currents for PEG3k compared to PEG5k are the result of a higher surface density

while a smaller fraction of the polymer chains is long enough to contribute to

electron transfer.

Figure 6.4: a) The effect of linker length on the obtained maximum surface density after 3 h

functionalization, and b) the corresponding redox cycling currents, presented as the rate of

electrons transferred per PEG chain. The measurements were performed on both types of devices,

Type I = , Type II = ᴏ.

When considering the mechanism by which the electrons are transferred, it was

anticipated that the polymer length of PEG10k is long enough to enable the

attached ferrocene moiety to travel to the opposing electrode, whereas the

chain length for PEG5k can only provide for electron transfer between the

ferrocene moieties itself inside the channel in the zone where layer

interpenetration and Fc-Fc electron transfer events between oppositely

immobilized chains occur. Not taking into account the dispersity of the

polymers, the length of 40 nm of PEG5k gives rise to a 15 nm exchange zone

Chapter 6

108

inside the 65 nm channel where the polymer chains from both electrodes can

overlap and the ferrocene moieties can exchange electrons. For the PEG10k

chains to reach the opposing electrodes they have to stretch outwards and

penetrate through the polymer layer resulting in an exchange zone of 65 nm

(the entire channel). Next to larger exchange zone by a factor 4, the

concentration of ferrocene inside the channel is on average a factor 2 lower for

PEG10k than for PEG5k due to the lower surface densities, which corresponds to

an overall expected higher activity of a factor 2 for PEG10k. Additionally, for PEG-

10k there is no need to stretch fully to reach an opposing PEG chain. Taking all

these factors into account, we attribute the observed 5-fold higher activity of

PEG10k compared to PEG5k to electron transfer dominated by exchange between

the diffusing and interpenetrating ferrocene moieties in both cases, rather than

direct transfer to the opposing electrode. We expect the same mechanism to

hold for PEG3k as well, when taking the dispersity into account. In this case, only

a fraction of the molecules is sufficiently long to allow some interpenetration of

the chain ends from opposing electrodes which, combined with the higher

surface density, leads to the observed 3 times lower activity compared to PEG5k.

When taking the polymer chain length dispersities into account for all polymers,

the interpenetration and exchange zones do not have sharply defined

boundaries, but the exchange extends outside of the given borders with

decreasing probabilities. Since proper electron transfer rate calculations would

have to take into account the polymer length distributions, probability

distributions of the distance between the Fc end group and the electrode,

collision frequencies, polymer surface densities, diffusion rates, and Fc-Fc and

Fc-electrode electron transfer rates, we did not attempt this here. From the

almost linear behavior observed for the electron transfer per Fc moiety for the

polymers studied here (Figure 6.4b) and the arguments given above, we

conclude that the interpenetration and Fc-Fc exchange model (as depicted in

Figure 6.1c) holds for all polymer lengths studied here.

The conformation of polymers grafted to the surface depends on the surface

density, with the polymers existing in a mushroom conformation at low surface

densities, where the polymers are isolated from their neighboring chains, and

becoming more brush-like with increasing surface density as the surface

concentration passes a critical surface density.34 In order to study a possible

effect of the PEG density and conformation regime on the redox cycling

currents, Type I devices were functionalized with different surface densities of


109

PEG10k, by changing the time the molecules were allowed to react with the

cystamine monolayer. The resulting redox cycling currents were plotted vs. the

incubation time, as shown in Figure 6.5. The surface densities, especially for

short reaction times, were too low to give clearly distinguishable peaks in the

cyclic voltammograms, and no reliable surface densities could be recorded.

Therefore the redox cycling currents were compared with surface densities

determined from experiments on macro electrodes. As can be seen in Figure

6.5a, the obtained surface densities on the macro electrodes reach a plateau

around 40 pmol cm-2, which is more than a factor 2 higher than the values

obtained inside the nanogap devices (Figure 6.4a). Therefore it is expected that

the surface densities inside the nanogap devices are still in the linear regime (left

side of Figure 6.5a) for all incubation times used, probably due to slower mass

transport compared to the macro electrodes. Indeed, it can be seen (Figure

6.5b) that the recorded redox cycling currents increase linearly with the

incubation time. It can thus be concluded that the increase in current with

increasing reaction times is mostly the result of the increase of ferrocene density

and not of a change in polymer conformation. Due to the bulky nature of the

PEG10k molecule, all used reaction times result in polymers present in a loose

brush conformation (the maximum surface density for the mushroom

configuration for PEG10k is calculated as 2 pmol cm-2),34 and no dramatic change

of behavior could be observed.

Figure 6.5: a) Surface densities on macro electrodes, and b) redox cycling currents in nanogap

devices for PEG10k as a function of the incubation time.

6.3 Conclusions

We have shown that surface-tethered PEG polymer chains with redox-active

end groups allow the transfer of electrons between the electrodes in nanogap

Chapter 6

110

devices in the absence of an electrochemical mediator. The primary condition

that needs to be met is that the polymer chains of the opposing electrodes can

interpenetrate so that oxidized and reduced ferrocene moieties can exchange

electrons. The activity per Fc unit, as defined by the average number of electrons

transferred per Fc moiety, is strongly dependent on the chain length, but not on

the chain density in the loose brush density range studied here. The chain length

effect is interpreted primarily by the increasing thickness of the channel zone in

which exchange can occur and the increasing fraction of the polymers that can

reach this exchange zone. The overall measured cycling currents are additionally

dependent on the surface density, which is lower for longer polymers and thus

counteracts to some extent the enhanced activities observed for the longer

PEGs. All polymer lengths studied here were found to obey this same

interpenetration and electron exchange model.

The findings of this study can contribute to an enhanced understanding and

improved performance of biosensing devices that use redox cycling in surface-

tethered receptor devices as the main amplification mechanism, such as in DNA

hairpin-like detection schemes. For example, if a probe DNA molecule that

allows hairpin formation is inserted in an immobilized polymer chain, binding of

a complementary target DNA can break up the hairpin leading to an apparent

lengthening of the polymer chain and thus to an increased layer penetration and

enhanced current. A single binding event is thus affecting the electron transfer

of many electrons, hence causing amplification. An increase in current, and with

that an increase in sensitivity, can be expected if the height of the nanochannel

is more finely tuned towards the length of the linker. At the same time, the

selectivity between bound and unbound receptors can be improved when the

polymer length distribution is more narrow and when the extended chain length

is close to half of the channel width, so that small changes in polymer stiffness

and length have large effects on the probabilities of chain interpenetration and

electron exchange.


Materials

Reagents and solvents were purchased from Sigma-Aldrich, high-purity water

(MilliQ) was used (Millipore, R = 18.2 MΩ). All bis-NHS-functionalized PEGs were

purchased from Nanocs and have a reported dispersity of 1.08. The Fc-PEG-NHS

molecules were synthesized according to known procedures.10 The obtained Fc-


111

PEG3k-NHS, Fc-PEG5k-NHS, and Fc-PEG10k-NHS was further purified by size

exclusion chromatography (Biobeads SX-1) with DCM as eluent. The synthesis of

the Fc-tethered adsorbates with different lengths of PEG is described in Chapter

5.

Device fabrication

A 4-inch Si wafer was isolated with 500 nm thick thermally grown SiO2. A Pt

bottom electrode, a Cr sacrificial layer and a Pt top electrode were then

consecutively deposited by electron-beam evaporation of 20 nm, 60 nm and 100

nm of metal, respectively, and patterned using a lift-off process based on a

positive photoresist (OIR 907-17, Arch Chemicals). Thereafter, a passivation

layer consisting of 90 nm / 325 nm / 90 nm thick PECVD SiO2/Si3N4/SiO2 was

deposited. Access holes were then etched through the passivation layer in by

reactive ion etching, reaching the Cr sacrificial layer. Lastly, the sacrificial layer

was etched by placing a drop of chromium etchant (BASF, Chromium Etch

Selectipur) at the inlets of the nanochannel. Additional details of the fabrication

process have been described elsewhere.36-37

Electrochemical experiments in the nanodevice

The electrodes were cleaned prior to the measurements by cycling their

potential in 0.5 M H2SO4 between -0.15 and 1.2 V at 50 mV/s until a stable

voltammogram was obtained. The devices were rinsed with MilliQ water and a

polydimethylsiloxane (PDMS) reservoir was positioned above the device which

and was filled with a 2 mM solution of cystamine and left overnight.

Subsequently, the devices were rinsed with a copious amount of water and filled

with a 0.2 mM Fc-PEG-NHS solution in water for 3 h or the incubation times

indicated in Figure 6.5, after which the device was immersed in a beaker with

MilliQ water which was vigorously stirred for at least 5 min (control

measurements have shown that this is sufficient time to remove all physisorbed

PEG chains from the channel, see Figure 6.6). Before measurements, the devices

were filled with a 1M NaClO4 supporting electrolyte solution, and a standard

Ag/AgCl electrode (BASi, MF 2079, RE-5B) inserted in the PDMS reservoir was

used as the reference electrode. No counter electrode was used as the current

through the reference is minimal in this configuration. Two transimpedance

amplifiers (Femto, DDPCA-300) were used to apply potentials to the two

Chapter 6

112

working electrodes with respect to the reference electrode and monitor the

currents through these electrodes.

Figure 6.6: Redox cycling currents of a nanodevice before (black) and after (red) rinsing in water,

after functionalization with PEG10k and 2-mercaptoethanol instead of cystamine. Measurements

were performed at a scan rate of 10 mV/s.

Assessment of electrode surface roughness

Devices were functionalized overnight with 2 mM 11-(ferrocenyl)undecanethiol

in ethanol. Due to the high surface densities and suppression of the capacity by

the long alkyl chains, surface densities could be determined for the top and

bottom electrodes separately. The surface densities of the top and bottom

electrodes were calculated as Γ = 1.9 and 0.59 nmol/cm2, respectively. Given

that the surface density of a full ferrocene layer is 0.45 nmol/cm2,1 and assuming

that both the electrodes are similarly functionalized, the determined surface

densities imply a higher surface roughness for the top electrode than the

bottom (4.2 and 1.3, respectively, for this particular device).

Macro electrode functionalization

The Fc-PEG surface densities at different incubation times were measured on

macro electrodes (2 mm diameter gold disc electrodes; CH Instruments). Before

modification the electrodes were polished using 50 nm alumina particles (CH

Instruments), followed by extensive rinsing with ethanol and 5 min of ultrasonic

treatment in ethanol and 5 min in MilliQ water. Subsequently the electrodes

were cleaned electrochemically in 0.5 M H2SO4 by applying an oxidizing potential


113

of 2 V for 5 s followed by a reducing potential of -0.35 V for 10 s. Then the

electrode potential was scanned from -0.25 V to 1.55 V and back for 40 cycles at

a scan rate of 100 mV/s. After cleaning the electrodes were rinsed with MilliQ

water and ethanol and dried under a flow of N2 and placed immediately in a 2

mM cystamine solution and left overnight. The surface densities of PEG were

varied by changing the incubation time with a 0.2 mM PEG solution and

determined from by cyclic voltammograms measured at scan rates between 50

and 100 mV/s, using a CH instruments bipotentiostat 760D, with 1 M NaClO4 as

electrolyte vs. a Ag/AgCl reference electrode and a platinum counter electrode.

6.5 References

(1) Hamburg, M. A.; Collins, F. S., The Path to Personalized Medicine. N. Engl. J. Med. 2010, 363,

301-304.

(2) Wang, J., Electrochemical Biosensors: Towards Point-of-Care Cancer Diagnostics. Biosens.

Bioelectron. 2006, 21, 1887-1892.

(3) Kirsch, J.; Siltanen, C.; Zhou, Q.; Revzin, A.; Simonian, A., Biosensor Technology: Recent

Advances in Threat Agent Detection and Medicine. Chem. Soc. Rev. 2013, 42, 8733-8768.

(4) Perumal, V.; Hashim, U., Advances in Biosensors: Principle, Architecture and Applications. J.

Appl. Biomed. 2014, 12, 1-15.

(5) Rackus, D. G.; Shamsi, M. H.; Wheeler, A. R., Electrochemistry, Biosensors and Microfluidics: A

Convergence of Fields. Chem. Soc. Rev. 2015, 44, 5320-5340.



A. 2003, 100, 9134-9137.

(7) Xiao, Y.; Qu, X.; Plaxco, K. W.; Heeger, A. J., Label-Free Electrochemical Detection of DNA in

Blood Serum Via Target-Induced Resolution of an Electrode-Bound DNA Pseudoknot. J. Am. Chem.

Soc. 2007, 129, 11896-11897.

(8) Xiao, Y.; Lou, X.; Uzawa, T.; Plakos, K. J. I.; Plaxco, K. W.; Soh, H. T., An Electrochemical Sensor

for Single Nucleotide Polymorphism Detection in Serum Based on a Triple-Stem DNA Probe. J. Am.

Chem. Soc. 2009, 131, 15311-15316.







Chapter 6

114

(11) Blauch, D. N.; Saveant, J. M., Dynamics of Electron Hopping in Assemblies of Redox Centers.

Percolation and Diffusion. J. Am. Chem. Soc. 1992, 114, 3323-3332.

(12) Anne, A.; Bouchardon, A.; Moiroux, J., 3′-Ferrocene-Labeled Oligonucleotide Chains End-

Tethered to Gold Electrode Surfaces: Novel Model Systems for Exploring Flexibility of Short DNA

Using Cyclic Voltammetry. J. Am. Chem. Soc. 2003, 125, 1112-1113.

(13) Huang, K. C.; White, R. J., Random Walk on a Leash: A Simple Single-Molecule Diffusion Model

for Surface-Tethered Redox Molecules with Flexible Linkers. J. Am. Chem. Soc. 2013, 135, 12808-

12817.



Eur. J. 2011, 17, 9678-9690.

(15) Smalley, J. F.; Feldberg, S. W.; Chidsey, C. E. D.; Linford, M. R.; Newton, M. D.; Liu, Y.-P., The

Kinetics of Electron Transfer through Ferrocene-Terminated Alkanethiol Monolayers on Gold. J.

Phys. Chem. 1995, 99, 13141-13149.

(16) Weber, K.; Hockett, L.; Creager, S., Long-Range Electronic Coupling between Ferrocene and

Gold in Alkanethiolate-Based Monolayers on Electrodes. J. Phys. Chem. B 1997, 101, 8286-8291.

(17) Finklea, H. O.; Hanshew, D. D., Electron-Transfer Kinetics in Organized Thiol Monolayers with

Attached Pentaammine(Pyridine)Ruthenium Redox Centers. J. Am. Chem. Soc. 1992, 114, 3173-

3181.

(18) Merchant, S. A.; Meredith, M. T.; Tran, T. O.; Brunski, D. B.; Johnson, M. B.; Glatzhofer, D. T.;

Schmidtke, D. W., Effect of Mediator Spacing on Electrochemical and Enzymatic Response of

Ferrocene Redox Polymers. J. Phys. Chem. C 2010, 114, 11627-11634.

(19) Zevenbergen, M. A. G.; Krapf, D.; Zuiddam, M. R.; Lemay, S. G., Mesoscopic Concentration

Fluctuations in a Fluidic Nanocavity Detected by Redox Cycling. Nano Lett. 2007, 7, 384-388.

(20) Zhao, C.; Wittstock, G., An Secm Detection Scheme with Improved Sensitivity and Lateral

Resolution: Detection of Galactosidase Activity with Signal Amplification by Glucose

Dehydrogenase. Angew. Chem. Int. Ed. 2004, 43, 4170-4172.

(21) Rassaei, L.; Mathwig, K.; Kang, S.; Heering, H. A.; Lemay, S. G., Integrated Biodetection in a

Nanofluidic Device. ACS Nano 2014, 8, 8278-8284.

(22) Abbou, J.; Anne, A.; Demaille, C., Probing the Structure and Dynamics of End-Grafted Flexible

Polymer Chain Layers by Combined Atomic Force-Electrochemical Microscopy. Cyclic

Voltammetry within Nanometer-Thick Macromolecular Poly(Ethylene Glycol) Layers. J. Am. Chem.

Soc. 2004, 126, 10095-10108.

(23) Wang, K.; Goyer, C.; Anne, A.; Demaille, C., Exploring the Motional Dynamics of End-Grafted

DNA Oligonucleotides by in Situ Electrochemical Atomic Force Microscopy. J. Phys. Chem. B 2007,

111, 6051-6058.

(24) Rassaei, L.; Singh, P. S.; Lemay, S. G., Lithography-Based Nanoelectrochemistry. Anal. Chem.

2011, 83, 3974-3980.


115

(25) Dam, V. A. T.; Olthuis, W.; van den Berg, A., Redox Cycling with Facing Interdigitated Array

Electrodes as a Method for Selective Detection of Redox Species. Analyst 2007, 132, 365-370.

(26) Goluch, E. D.; Wolfrum, B.; Singh, P. S.; Zevenbergen, M. A. G.; Lemay, S. G., Redox Cycling in

Nanofluidic Channels Using Interdigitated Electrodes. Anal. Bioanal. Chem. 2009, 394, 447-456.

(27) Samarao, A. K.; Rust, M. J.; Ahn, C. H. In Rapid Fabrication of a Nano Interdigitated Array

Electrode and Its Amperometric Characterization as an Electrochemical Sensor, Proc. IEEE Sens.,

2007; pp 644-647.

(28) Ma, C.; Contento, N. M.; Gibson, L. R.; Bohn, P. W., Redox Cycling in Nanoscale-Recessed Ring-

Disk Electrode Arrays for Enhanced Electrochemical Sensitivity. ACS Nano 2013, 7, 5483-5490.

(29) Kätelhön, E.; Hofmann, B.; Lemay, S. G.; Zevenbergen, M. A. G.; Offenhäusser, A.; Wolfrum,

B., Nanocavity Redox Cycling Sensors for the Detection of Dopamine Fluctuations in Microfluidic

Gradients. Anal. Chem. 2010, 82, 8502-8509.

(30) Xiong, J.; Chen, Q.; Edwards, M. A.; White, H. S., Ion Transport within High Electric Fields in

Nanogap Electrochemical Cells. ACS Nano 2015, 9, 8520-8529.

(31) For the determination of the surface densities, the electrodes could not be cycled individually

as the two electrodes are capacitively coupled, giving rise to a situation analogous to the positive-

feedback mode of SECM with an unbiased electrode. See: Oleinick, A. I.; Battistel, D.; Daniele, S.;

Svir, I.; Amatore, C., Simple and Clear Evidence for Positive Feedback Limitation by Bipolar

Behavior During Scanning Electrochemical Microscopy of Unbiased Conductors. Anal. Chem. 2011,

83, 4887-4893.



(33) Allen, C.; Dos Santos, N.; Gallagher, R.; Chiu, G. N. C.; Shu, Y.; Li, W. M.; Johnstone, S. A.;

Janoff, A. S.; Mayer, L. D.; Webb, M. S.; Bally, M. B., Controlling the Physical Behavior and Biological

Performance of Liposome Formulations through Use of Surface Grafted Poly(Ethylene Glycol).

Biosci. Rep. 2002, 22, 225-250.

(34) De Gennes, P. G., Conformations of Polymers Attached to an Interface. Macromolecules 1980,

13, 1069-1075.



1485-1492.

(36) Zevenbergen, M. A. G.; Wolfrum, B. L.; Goluch, E. D.; Singh, P. S.; Lemay, S. G., Fast Electron-

Transfer Kinetics Probed in Nanofluidic Channels. J. Am. Chem. Soc. 2009, 131, 11471-11477.

(37) Kang, S.; Mathwig, K.; Lemay, S. G., Response Time of Nanofluidic Electrochemical Sensors.

Lab Chip 2012, 12, 1262-1267.

Chapter 6

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117

Summary

Electron transfer processes play an important role in the development of

nanoelectronics and biosensors. Therefore it is important to understand and to

optimize these processes. Self-assembled monolayers (SAMs) provide a highly

tunable platform which can be implemented on metallic electrodes for the use

of electrochemical techniques. The research described in this thesis considers

two different types of monolayer surfaces with the aim to tune and study their

electron transfer properties.

Chapter 2 provides a general overview of several electrochemical techniques

and how they can be used in the determination of electron transfer rate

constants. A further focus is on flexible linear probes such as DNA for the use in

sensors, in which their flexibility is altered upon binding of a complementary

sequence and the resulting conformational changes affect the signal output.

In the first part of this thesis (Chapters 3 and 4) six multivalent β-cyclodextrin (β-

CD) adsorbates have been described. The effect of the different functional

groups on the adsorption kinetics, thickness, layer stability has been evaluated

(Chapter 3). Even weakly binding anchoring groups gave rise to rather stable

monolayers and several of these compounds were selected for further study.

These novel β-CD adsorbates were designed with their cavity in close contact to

the gold electrode. The effect of this small distance on the electron transfer

rates, when compared to placing the β-CD core at a distance from the surface

by long alkyl tethers, was studied using different electrochemical probes

(Chapter 4). Decreasing the distance between the β-CD core and the underlying

substrate led to an increase in the electron transfer rates.

In the second part of this thesis (Chapters 5 and 6), the electron transfer to/from

ferrocene (Fc) moieties tethered to long linear poly(ethylene glycol) (PEG)

molecules attached to a gold electrode was studied. In Chapter 5, the Fc-PEGs

were attached to macro electrodes. The electron transfer to/from the Fc

moieties was shown to be governed by bounded diffusion, and the effects of

conformational changes by means of tether length and surface density were

probed. The introduction of the Fc-PEG molecules between two nanospaced

electrodes (Chapter 6) showed that the bounded diffusion made it possible to

118

shuttle electrons between two electrodes. This provides a powerful mechanism

for signal-enhanced biosensor devices.

The results described in this thesis show that the versatility of SAMs makes it

possible to tune the properties of these layers to enhance or modify the electron

transfer properties. Electrochemistry provides a powerful tool for the

characterization of these layers. The results presented here provide a

contribution to the development of electrochemical biosensing devices.

119

Samenvatting

Elektronenoverdrachtsprocessen spelen een belangrijke rol in de ontwikkeling

van nanoelektronica en biosensoren. Daarom is het belangrijk om deze

processen te begrijpen en te optimaliseren. Zelf-assemblerende monolagen

(SAMs) vormen een veelzijdig platform dat toegepast kan worden op

metallische elektroden voor het gebruik van elektrochemische technieken. Het

in dit proefschrift beschreven onderzoek beschouwt twee verschillende typen

monolaagoppervlakken met als doel de elektronoverdrachtseigenschappen te

kunnen controleren en te bestuderen.

Hoofdstuk 2 geeft een algemeen overzicht van verschillende elektrochemische

technieken en hoe deze gebruikt kunnen in de bepaling van

elektronenoverdrachts-constanten. Verder ligt er een focus op flexibele lineaire

moleculen zoals DNA voor het gebruik in sensoren, waarin de flexibiliteit

verandert na binding met een complementaire sequentie. De resulterende

conformationele veranderingen beïnvloeden daardoor het uitgangssignaal.

In het eerste deel van dit proefschrift (hoofdstukken 3 en 4) worden zes

multivalente β-cyclodextrine- (β-CD-)adsorbaten beschreven. Het effect van de

verschillende functionele groepen op de adsorptiekinetiek, laagdikte en

stabiliteit is geëvalueerd (hoofdstuk 3). Zelfs zwak bindende functionele

groepen bleken een redelijk stabiele monolaag te geven en een aantal van de

ontwikkelde β-CD-adsorbaten werden geselecteerd voor verdere studie. Deze

nieuwe β-CD-adsorbaten zijn ontworpen zodat hun holte vlakbij de goud-

elektrode gepositioneerd kon worden. Het effect van deze kleine afstand op de

elektronoverdrachtssnelheid, vergeleken met een β-CD-holte op afstand van

het oppervlak gefaciliteerd door lange alkylketens, werd bestudeerd door

middel van verschillende elektrochemische sondes (hoofdstuk 4). Het

verminderen van de afstand tussen de β-CD-kern en het onderliggende

substraat veroorzaakte een stijging in de elektronenoverdrachtssnelheid.

In het tweede deel van dit proefschrift (hoofdstukken 5 en 6) werd de

elektronenoverdracht van en naar ferroceen- (Fc-)groepen, vastgezet op goud-

elektroden via lange lineaire poly(ethyleenglycol)- (PEG-)moleculen,

bestudeerd. In hoofdstuk 5 werden de Fc-PEGs vastgezet aan macro-elektroden.

De elektronenoverdracht van en naar de Fc-groepen werd gestuurd door

120

gebonden diffusie, en de effecten van conformationele veranderingen aan de

hand van het aanpassen van de PEG-lengte en de oppervlaktedichtheid werden

bestudeerd. Het aanbrengen van de Fc-PEG-moleculen tussen twee elektroden

met een onderlinge afstand van 65 nm (hoofdstuk 6) liet zien dat de gebonden

diffusie het mogelijk maakte om elektronen tussen de twee elektroden te laten

bewegen. Dit biedt een krachtig mechanisme voor signaalversterkende

biosensor-apparaten.

De in dit proefschrift beschreven resultaten laten zien dat de veelzijdigheid van

SAMs het mogelijk maakt de eigenschappen van deze lagen aan te passen om

de elektronenoverdrachtseigenschappen te versterken of aan te passen.

Elektrochemie biedt krachtige methodes voor de karakterisering van deze lagen.

De hier beschreven resultaten leveren een bijdrage aan de ontwikkeling van

elektrochemische biosensor-apparaten.

121

Acknowledgements

And so we find ourselves at the end of this thesis, the result of several years of

work. Now that the end of my PhD is near and my defense is only a few weeks

away it is time to reflect a bit on the past years and give credit where credit is

due. Because, without a doubt none of this would have been possible without

the help and support of a large number of people.

To start at the top, special thanks go out to my promotors Jurriaan en Pascal

who gave me the chance to work in MnF. Thanks to you both for being inspiring

and for putting me back on track when I felt a bit stuck and helping me get my

first grasp on electrochemistry. You both played a big part in me pursuing this

path, Pascal having been my supervisor since my master project and Jurriaan

going all the way back to my bachelor project.

A lot of our early experiments were performed in the BIOS labs, thanks to Albert

van den Berg for the opportunity to be part of the ELab4Life project. Maarten,

you were my first collaborator, it’s a shame that our devices never turned out

to be useful for the purposes we had in mind, but I’m glad you got some good

results out of them for a different project. Whether you liked it or not, you also

became my resident expert on all things related to electrochemistry. Thanks for

the many, many fruitful discussions and sharing my frustrations when we

started figuring out how to measure on electrodes.

The answer to all our device problems came from the NanoIonics group. Serge,

many thanks for the opportunity to do measurements in your group and the

discussions we’ve had and of course for being part of my committee. Sahana,

we’ve spend a lot of time in the lab doing late night/weekend measurements on

your devices. Not all measurements were equally useful, but after the discovery

of some sort of magical washing step everything fell into place and led to a nice

paper.

From within our own group, Alejandro, without your synthesis of all the

cyclodextrin varieties and probes and many other inputs, half of this thesis

would have been impossible. I’m very grateful for all your help and

contributions. For these chapters, the contributions and discussions with

122

Bernard Boukamp were also of great value and of course thanks for being part

of my committee.

Many thanks scientific staff of MnF/BNT, Wim, Tibor, Jeroen (also for being part

of my committee) and Nathalie for all their input during colloquia and other

forms of advice over the years. Nicole and Izabel for their help with all

paperwork related issues. Of course the technical staff: Richard, Marcel, Regine

and Bianca for keeping the labs running smoothly, and their company during all

those coffee breaks together with the rest of the “coffee group”.

Many thanks to all MnF/BNT PhDs and postdocs that passed through the labs in

all those years for the great working atmosphere, group outings, work weeks

and so forth, the list is much too long to name all of you. Special thanks to the

people of lab 4, where I spend most of my measuring time (in darkness, literally,

not metaphorically). Thanks to all members of the Assembly and Biomolecules

meetings for their input and discussions, and for putting up with all my

ramblings about electrochemistry.

Tot slot wil ik graag familie en vrienden bedanken, die mij op allerlei manieren

geholpen hebben. Mijn huisgenoten voor de nodige ontspanning in de

avonduren. De leden van de Opbloasband voor de nodige muzikale ontspanning

in de weekenden. De vriendengroep die het al sinds de middelbare school met

elkaar uithoudt, we zien elkaar niet zo vaak als vroeger door de familiaire

uitbreidingen en drukke agenda’s, maar het is nog altijd gezellig en de sfeer is

nog altijd goed. Mijn zusjes, Lindy en Laura, voor hun steun en hun

bereidwilligheid om mijn paranimfen te zijn (“Nee, dat doen wij”, waren volgens

mij de exacte woorden), hun wederhelften Alan en Tommy en natuurlijk de

kleine Pim en Fiene. Als allerlaatste gaat mijn grote dank uit naar mijn ouders

voor hun steun door de jaren heen.

Again, many thanks to everyone who was part of this journey and for the fond

memories and experiences!

Tom

123

Curriculum Vitae

Tom Steentjes was born on the 18th of October 1982 in Doetinchem, the

Netherlands. He studied Chemical Engineering at the University of Twente in

Enschede, the Netherlands, where he received his Bachelor degree in 2007. His

Bachelor thesis was entitled “Surface diffusion of two divalent molecular inks on

a molecular printboard” and was performed in the Supramolecular Chemistry

and Technology group under the supervision of prof. dr. ir. Jurriaan Huskens.

In December 2010 he received his MSc degree in Chemical Engineering. His

internship, entitled “Immobilization of quantum dots on a functionalized

surface”, was carried out at the Institute of Materials Research and Engineering

in Singapore, under the supervision of prof. dr. G.J. Vancso and dr. N. Tomczak.

His master thesis, entitled “Light-induced immobilization of a novel perylene

bisimide by the thiol-ene click reaction”, was performed in the Molecular

NanoFabrication group under the supervision of prof. dr. ir. Jurriaan Huskens

and prof. dr. ir. Pascal jonkheijm.

Since January 2011 he has been working as a PhD candidate in the Molecular

NanoFabrication group under the supervision of prof. dr. ir. Jurriaan Huskens

and prof. dr. ir. Pascal Jonkheijm. The results of this research are described in

this thesis.

Documents

Self-assembled monolayers probed by sensorsmonolayer surfaces. First, the design and characterization of several novel β-cyclodextrin (β-CD) adsorbates is presented (Chapters 3 and